Science.gov

Sample records for mitochondrial apoptotic pathways

  1. Genes of the Mitochondrial Apoptotic Pathway in Mytilus galloprovincialis

    PubMed Central

    Figueras, Antonio; Novoa, Beatriz

    2013-01-01

    Bivalves play vital roles in marine, brackish, freshwater and terrestrial habitats. In recent years, these ecosystems have become affected through anthropogenic activities. The ecological success of marine bivalves is based on the ability to modify their physiological functions in response to environmental changes. One of the most important mechanisms involved in adaptive responses to environmental and biological stresses is apoptosis, which has been scarcely studied in mollusks, although the final consequence of this process, DNA fragmentation, has been frequently used for pollution monitoring. Environmental stressors induce apoptosis in molluscan cells via an intrinsic pathway. Many of the proteins involved in vertebrate apoptosis have been recognized in model invertebrates; however, this process might not be universally conserved. Mytilus galloprovincialis is presented here as a new model to study the linkage between molecular mechanisms that mediate apoptosis and marine bivalve ecological adaptations. Therefore, it is strictly necessary to identify the key elements involved in bivalve apoptosis. In the present study, six mitochondrial apoptotic-related genes were characterized, and their gene expression profiles following UV irradiation were evaluated. This is the first step for the development of potential biomarkers to assess the biological responses of marine organisms to stress. The results confirmed that apoptosis and, more specifically, the expression of the genes involved in this process can be used to assess the biological responses of marine organisms to stress. PMID:23626691

  2. MicroRNAs regulate mitochondrial apoptotic pathway in myocardial ischemia-reperfusion-injury.

    PubMed

    Makhdoumi, Pouran; Roohbakhsh, Ali; Karimi, Gholamreza

    2016-12-01

    MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional gene regulators. They are involved in the pathogenesis of different disorders including heart diseases. MiRNAs contribute to ischemia/reperfusion injury (I/RI) by altering numerous key signaling elements. Together with alterations in the various potential signaling pathways, modification in miRNA expression has been suggested as a part of the response network following ischemia/reperfusion (I/R). In addition, cardiac mitochondrial homeostasis is closely associated with cardiac function and impairment of mitochondrial activity occurred after ischemia/reperfusion injury. MiRNAs play a key role in the regulation of mitochondrial apoptotic pathway and signaling proteins. In this review, we summarize the knowledge currently available regarding the molecular mechanisms of miRNA-regulated mitochondrial functions during ischemia/reperfusion injury. This regulation occurs in different stages of mitochondrial apoptosis pathway.

  3. The Opa1-Dependent Mitochondrial Cristae Remodeling Pathway Controls Atrophic, Apoptotic, and Ischemic Tissue Damage

    PubMed Central

    Varanita, Tatiana; Soriano, Maria Eugenia; Romanello, Vanina; Zaglia, Tania; Quintana-Cabrera, Rubén; Semenzato, Martina; Menabò, Roberta; Costa, Veronica; Civiletto, Gabriele; Pesce, Paola; Viscomi, Carlo; Zeviani, Massimo; Di Lisa, Fabio; Mongillo, Marco; Sandri, Marco; Scorrano, Luca

    2015-01-01

    Summary Mitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo. PMID:26039448

  4. Chlorpromazine inhibits mitochondrial apoptotic pathway via increasing expression of tissue factor.

    PubMed

    Wu, Jing; Li, Aimei; Li, Yujun; Li, Xiaoguang; Zhang, Qingmeng; Song, Wuqi; Wang, Yao; Ogutu, James O; Wang, Jindong; Li, Jianbo; Tang, Renkuan; Zhang, Fengmin

    2016-01-01

    Chlorpromazine (CPZ) is a widely used antipsychotic drug with antagonistic effect on dopamine receptors. Accumulating evidence has shown that CPZ plays a neuroprotective role in various models of toxicity and apoptosis. However, the underlying mechanism contributing to this protective effect remains unclear. Here, we evaluate the effect of CPZ on mitochondrial apoptotic pathway in the neuron system. Higher levels of B-cell lymphoma-2 (Bcl-2) and tissue factor (TF) but lower apoptotic rate were found in hippocampus of CPZ-treated schizophrenic patients compared with non-antipsychotic treated controls. Additionally, both short-term and long-term treatment of CPZ in rats could up-regulate the levels of Bcl-2 and TF with no cytotoxic effects. In the in vitro experiment, expression of Bcl-2 was up-regulated in the C6 glioma cells transfected with pEGFP-N1-TF recombinant plasmid. Furthermore, in another independent rat model of apoptosis, compared with the group administrated with alcohol only, the brains of the CPZ-pretreated rats showed lower expression of cleaved caspase-3, cytochrome c and Bax, but higher expression of Bcl-2 and TF. Our data demonstrate that CPZ exerts its neuronal protective effects through inhibiting the activation of mitochondrial apoptotic pathway by up-regulating TF expression, thus providing new insight into CPZ function and application.

  5. Galectin-1 and Galectin-3 induce mitochondrial apoptotic pathway in Jurkat cells

    NASA Astrophysics Data System (ADS)

    Vasil'eva, O. A.; Isaeva, A. V.; Prokhorenko, T. S.; Zima, A. P.; Novitsky, V. V.

    2016-08-01

    Cellular malignant transformation is often accompanied by increased gene expression of low-molecular proteins of lectins family-galectins. But it is unknown how galectins promote tumor growth and malignization. Galectins-1 and galectin-3 are thought to be possible immunoregulators exerting their effects by regulating the balance of CD4+ lymphocytes. In addition it is known that tumor cells overexpressing galectins are capable of escaping immunological control, causing apoptosis of lymphocytes. The aim of the study is to investigate the role of galectin-1 and galectin-3 in the implementation of mitochondrial apoptotic pathway in Jurkat cells. Methods: Jurkat cells were used as a model for the study of T-lymphocytes. Jurkat cells were activated with antibodies to CD3 and CD28 and cultured with recombinant galectin-1 and -3. Apoptosis of Jurkat cells and depolarization of the mitochondrial membrane were assessed by flow cytometry. It was found that galectin-1 and galectin-3 have a dose-dependent pro-apoptotic effect on Jurkat cells in vitro and enlarge the number of cells with decreased mitochondrial membrane potential compared with intact cells.

  6. Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer's disease

    PubMed Central

    Wang, Yan; Cai, Biao; Shao, Jing; Wang, Ting-ting; Cai, Run-ze; Ma, Chang-ju; Han, Tao; Du, Jun

    2016-01-01

    Genistein is effective against amyloid-β toxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer’s disease. A rat model of Alzheimer’s disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-β peptide (25–35). In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg) was given prior to establishing Alzheimer’s disease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genistein in Alzheimer’s disease rats. These findings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c. PMID:27630702

  7. Iron overload induced death of osteoblasts in vitro: involvement of the mitochondrial apoptotic pathway

    PubMed Central

    Dai, Zhipeng; Yang, Jingjing; Zheng, Jin

    2016-01-01

    Background Iron overload is recognized as a new pathogenfor osteoporosis. Various studies demonstrated that iron overload could induce apoptosis in osteoblasts and osteoporosis in vivo. However, the exact molecular mechanisms involved in the iron overload-mediated induction of apoptosis in osteoblasts has not been explored. Purpose In this study, we attempted to determine whether the mitochondrial apoptotic pathway is involved in iron-induced osteoblastic cell death and to investigate the beneficial effect of N-acetyl-cysteine (NAC) in iron-induced cytotoxicity. Methods The MC3T3-E1 osteoblastic cell line was treated with various concentrations of ferric ion in the absence or presence of NAC, and intracellular iron, cell viability, reactive oxygen species, functionand morphology changes of mitochondria and mitochondrial apoptosis related key indicators were detected by commercial kits. In addition, to further explain potential mechanisms underlying iron overload-related osteoporosis, we also assessed cell viability, apoptosis, and osteogenic differentiation potential in bone marrow-derived mesenchymal stemcells(MSCs) by commercial kits. Results Ferric ion demonstrated concentration-dependent cytotoxic effects on osteoblasts. After incubation with iron, an elevation of intracelluar labile iron levels and a concomitant over-generation of reactive oxygen species (ROS) were detected by flow cytometry in osteoblasts. Nox4 (NADPH oxidase 4), an important ROS producer, was also evaluated by western blot. Apoptosis, which was evaluated by Annexin V/propidium iodide staining, Hoechst 33258 staining, and the activation of caspase-3, was detected after exposure to iron. Iron contributed to the permeabilizatio of mitochondria, leading to the release of cytochrome C (cyto C), which, in turn, induced mitochondrial apoptosis in osteoblasts via activation of Caspase-3, up-regulation of Bax, and down-regulation of Bcl-2. NAC could reverse iron-mediated mitochondrial dysfunction and

  8. Mitochondrial ROS and the Effectors of the Intrinsic Apoptotic Pathway in Aging Cells: The Discerning Killers!

    PubMed Central

    Hekimi, Siegfried; Wang, Ying; Noë, Alycia

    2016-01-01

    It has become clear that mitochondrial reactive oxygen species (mtROS) are not simply villains and mitochondria the hapless targets of their attacks. Rather, it appears that mitochondrial dysfunction itself and the signaling function of mtROS can have positive effects on lifespan, helping to extend longevity. If events in the mitochondria can lead to better cellular homeostasis and better survival of the organism in ways beyond providing ATP and biosynthetic products, we can conjecture that they act on other cellular components through appropriate signaling pathways. We describe recent advances in a variety of species which promoted our understanding of how changes of mtROS generation are part of a system of signaling pathways that emanate from the mitochondria to impact organism lifespan through global changes, including in transcriptional patterns. In unraveling this, many old players in cellular homeostasis were encountered. Among these, maybe most strikingly, is the intrinsic apoptotic signaling pathway, which is the conduit by which at least one class of mtROS exercise their actions in the nematode Caenorhabditis elegans. This is a pathway that normally contributes to organismal homeostasis by killing defective or otherwise unwanted cells, and whose various compounds have also been implicated in other cellular processes. However, it was a surprise that that appropriate activation of a cell killing pathway can in fact prolong the lifespan of the organism. In the soma of adult C. elegans, all cells are post-mitotic, like many of our neurons and possibly some of our immune cells. These cells cannot simply be killed and replaced when showing signs of dysfunction. Thus, we speculate that it is the ability of the apoptotic pathway to pull together information about the functional and structural integrity of different cellular compartments that is the key property for why this pathway is used to decide when to boost defensive and repair processes in irreplaceable

  9. Tetramethylpyrazine Protects against Early Brain Injury after Experimental Subarachnoid Hemorrhage by Affecting Mitochondrial-Dependent Caspase-3 Apoptotic Pathway

    PubMed Central

    Xiao, Xiaolan

    2017-01-01

    This study was to test the hypothesis that tetramethylpyrazine (TMP) protected against early brain injury after subarachnoid hemorrhage (SAH) by affecting the mitochondrial-dependent caspase-3 apoptotic pathway. TMP was administrated after the rats' prechiasmatic SAH mode. Animal neurobehavioral functions were assessed and the mitochondrial morphology, mitochondrial and cytoplasmic calcium, and mitochondrial membrane potential changes (Δψm) of the brain tissues were measured. The expressions of cytoplasmic cytochrome c (cyt c), second mitochondria-derived activator of caspases (Smac), and cleaved caspase-3 B-cell lymphoma 2 (bcl-2) in cells were determined and cellular apoptosis was detected. The treatment of TMP resulted in less apoptotic cells and milder mitochondrial injury and potentially performed better in the neurobehavioral outcome compared to those with saline. Also, TMP ameliorated calcium overload in mitochondria and cytoplasm and alleviated the decrease of Δψm. In addition, TMP inhibited the expression of cytoplasmic cyt c, Smac, and cleaved caspase-3, yet it upregulated the expression of bcl-2. These findings suggest that TMP exerts an antiapoptosis property in the SAH rat model and this is probably mediated by the caspase-3 apoptotic pathway triggered by mitochondrial calcium overload. The finding offers a new therapeutic candidate for early brain injury after SAH. PMID:28337226

  10. The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells.

    PubMed

    Liang, Wei-Zhe; Chou, Chiang-Ting; Hsu, Shu-Shong; Liao, Wei-Chuan; Shieh, Pochuen; Kuo, Daih-Huang; Tseng, Hui-Wen; Kuo, Chun-Chi; Jan, Chung-Ren

    2015-01-05

    Eugenol, a natural phenolic constituent of clove oil, has a wide range of applications in medicine as a local antiseptic and anesthetic. However, the effect of eugenol on human glioblastoma is unclear. This study examined whether eugenol elevated intracellular free Ca(2+) levels ([Ca(2+)]i) and induced apoptosis in DBTRG-05MG human glioblastoma cells. Eugenol evoked [Ca(2+)]i rises which were reduced by removing extracellular Ca(2+). Eugenol-induced [Ca(2+)]i rises were not altered by store-operated Ca(2+) channel blockers but were inhibited by the PKC inhibitor GF109203X and the transient receptor potential channel melastatin 8 (TRPM8) antagonist capsazepine. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished eugenol-induced [Ca(2+)]i rises. The phospholipase C (PLC) inhibitor U73122 significantly inhibited eugenol-induced [Ca(2+)]i rises. Eugenol killed cells which were not reversed by prechelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Eugenol induced apoptosis through increasing reactive oxygen species (ROS) production, decreasing mitochondrial membrane potential, releasing cytochrome c and activating caspase-9/caspase-3. Together, in DBTRG-05MG cells, eugenol evoked [Ca(2+)]i rises by inducing PLC-dependent release of Ca(2+) from the endoplasmic reticulum and caused Ca(2+) influx possibly through TRPM8 or PKC-sensitive channels. Furthermore, eugenol induced the mitochondrial apoptotic pathway.

  11. The apoptotic effect of apigenin on human gastric carcinoma cells through mitochondrial signal pathway.

    PubMed

    Chen, Jiayu; Chen, Jiaqi; Li, Zhaoyun; Liu, Chibo; Yin, Lihui

    2014-08-01

    This study aims to explore the apoptotic function of apigenin on the gastric cancer cells and the related mechanism. The gastric cancer cell lines HGC-27 and SGC-7901, and normal gastric epithelial cell line GES1 were treated with different concentrations of apigenin. Cell proliferation was tested. Morphological changes of the apoptotic cells were observed after Hoechst33342 staining. The apoptosis rate of the gastric cancer cells were measured with flow cytometry. Changes of the cell cycle were explored. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Bcl-2 family proteins and caspases-3 expression with apigenin treatment was analyzed by real-time PCR. Cell proliferation of HGC-27 and SGC-7901 was inhibited by apigenin, and the inhibition was dose-time-dependent. Gastric carcinoma cells treated by apigenin had no obvious cell cycle arrest, but were observed with the higher apoptosis rate and the typical apoptotic morphological changes of the cell nucleus. JC-1 staining showed that apigenin could reduce mitochondrial membrane potential of gastric carcinoma cells. Real-time PCR results showed that apigenin significantly increased caspase-3 and Bax expression level, and down-regulated Bcl-2 expression in a dose-dependent manner in gastric carcinoma cells. However, the GES1 was almost not affected by apigenin treatment. Apigenin can inhibit cell lines HGC-27 and SGC-7901 proliferation in a time and dose-dependent manner, reduce anti-apoptotic protein Bcl-2 levels, enhance apoptosis-promoting protein Bax level, result in mitochondrial membrane potential decreasing and caspase-3 enzyme activating, then lead to cell apoptosis.

  12. The canonical intrinsic mitochondrial death pathway has a non-apoptotic role in signaling lens cell differentiation.

    PubMed

    Weber, Gregory F; Menko, A Sue

    2005-06-10

    The mitochondrial cell death pathway is known for its role in signaling apoptosis. Here, we describe a novel function for the mitochondrial cell death pathway in signaling initiation of differentiation in the developing lens. Most remarkably, we induced lens cell differentiation by short-term exposure of lens epithelial cells to the apoptogen staurosporine. Activation of apoptosis-related pathways induced lens epithelial cells to express differentiation-specific markers and to undergo morphogenetic changes that led to formation of the lens-like structures known as lentoids. The fact that multiple stages of differentiation are expressed at a single stage of development in the embryonic lens made it possible to precisely determine the timing of expression of proteins associated with the apoptotic pathway. We discovered that there was high expression in the lens equatorial epithelium (the region of the lens in which differentiation is initiated) of pro-apoptotic molecules such as Bax and Bcl-x(S) and release of cytochrome c from mitochondria. Furthermore, we found significant caspase-3-like activity in the equatorial epithelium, yet this activity was far lower than that associated with lens cell apoptosis. These apoptotic pathways are likely regulated by the concurrent expression of prosurvival molecules, including Bcl-2 and Bcl-x(L); phosphorylation of Bad; and high expression of inhibitor of apoptosis proteins chicken IAP1, IAP3, and survivin. This finding suggests that prosurvival pathways allow pro-apoptotic molecules to function as molecular switches in the differentiation process without tipping the balance toward apoptosis. We call this process apoptosis-related Bcl-2- and caspase-dependent (ABC) differentiation.

  13. Induction of mitochondrial apoptotic pathway by raloxifene and estrogen in human endometrial stromal ThESC cell line

    PubMed Central

    Andjelkovic, Marija; Zaric, Milan; Zelen, Ivanka; Canovic, Petar; Milosavljevic, Zoran; Mitrovic, Marina

    2016-01-01

    Introduction Endometrial hyperplasia is a condition that occurs as a result of hormonal imbalance between estrogen and progesterone. Morphological disturbance of endometrial cells occurs consequently leading towards endometrial cancer. In therapy of endometrial hyperplasia SERMs are used to supress effects of locally high estrogen level in uterus. There is strong evidence suggesting that estrogen could be involved in cell death – apoptosis. There are no experimental data demstrating the direct apoptotic effect of both raloxifene and estrogen on the ThESC cell line. The aim of our study wa sto investigate both cytotoxic and apototic mechanism of raloxifene and estrogen – induced death in the ThESC cell line. Material and methods In order to determine their cytotoxic and apoptotic effects, various doses of raloxifene and estrogen were applied to the ThESC cell line for 24 h. After the treatment MTT assay, FACS analysis and immunofluoroscence method were conducted. Results The results of this study for the first time demonstrated the cytotoxic and apoptotic effects of raloxifene and estrogen on human endometrial stromal cell line suggesting the involvement of the inner, mitochondrial apoptotic pathway. Conclusions Our results demonstrated apoptotic effects of investigated drugs in the ThESC cell line through increasing the Bax/Bcl-2 ratio and activation of caspase 3. PMID:28261281

  14. Sequence and expression variations in 23 genes involved in mitochondrial and non-mitochondrial apoptotic pathways and risk of oral leukoplakia and cancer.

    PubMed

    Datta, Sayantan; Ray, Anindita; Singh, Richa; Mondal, Pinaki; Basu, Analabha; De Sarkar, Navonil; Majumder, Mousumi; Maiti, Guruparasad; Baral, Aradhita; Jha, Ganga Nath; Mukhopadhyay, Indranil; Panda, Chinmay; Chowdhury, Shantanu; Ghosh, Saurabh; Roychoudhury, Susanta; Roy, Bidyut

    2015-11-01

    Oral cancer is usually preceded by pre-cancerous lesion and related to tobacco abuse. Tobacco carcinogens damage DNA and cells harboring such damaged DNA normally undergo apoptotic death, but cancer cells are exceptionally resistant to apoptosis. Here we studied association between sequence and expression variations in apoptotic pathway genes and risk of oral cancer and precancer. Ninety nine tag SNPs in 23 genes, involved in mitochondrial and non-mitochondrial apoptotic pathways, were genotyped in 525 cancer and 253 leukoplakia patients and 538 healthy controls using Illumina Golden Gate assay. Six SNPs (rs1473418 at BCL2; rs1950252 at BCL2L2; rs8190315 at BID; rs511044 at CASP1; rs2227310 at CASP7 and rs13010627 at CASP10) significantly modified risk of oral cancer but SNPs only at BCL2, CASP1and CASP10 modulated risk of leukoplakia. Combination of SNPs showed a steep increase in risk of cancer with increase in "effective" number of risk alleles. In silico analysis of published data set and our unpublished RNAseq data suggest that change in expression of BID and CASP7 may have affected risk of cancer. In conclusion, three SNPs, rs1473418 in BCL2, rs1950252 in BCL2L2 and rs511044 in CASP1, are being implicated for the first time in oral cancer. Since SNPs at BCL2, CASP1 and CASP10 modulated risk of both leukoplakia and cancer, so, they should be studied in more details for possible biomarkers in transition of leukoplakia to cancer. This study also implies importance of mitochondrial apoptotic pathway gene (such as BCL2) in progression of leukoplakia to oral cancer.

  15. Taiwanofungus camphoratus (Syn Antrodia camphorata) extract and amphotericin B exert adjuvant effects via mitochondrial apoptotic pathway.

    PubMed

    Chen, Ling-Yi; Sheu, Ming-Thau; Liao, Chuh-Kai; Tsai, Feng-Chou; Kao, Woei-Yao; Su, Ching-Hua

    2013-03-01

    The use of multiple drugs in cancer therapy increases the efficacy of the potential therapeutic effects. In this study, the authors investigated the adjuvant effects of an ethanol extract of solid-state cultivated Taiwanofungus camphoratus (TCEE) and amphotericin B (AmB) in the human cancer cell lines RPMI7951 and MG63. Taiwanofungus camphoratus is a well-known Chinese medicine in Taiwan, and AmB is a widely used antifungal agent. The authors demonstrated that TCEE pretreatment followed by AmB treatment effectively inhibited cell growth. The combination of sublethal doses of TCEE and AmB revealed a significant growth inhibitory effect in both cell lines. The combination of TCEE and AmB but not AmB alone induced phosphatidylserine externalization and loss of mitochondrial membrane potential. Cell cycle analyses revealed that combination of TCEE and AmB triggered G2/M arrest and significant apoptosis after 48 hours. These effects were greater than those achieved using TCEE or AmB alone. Furthermore, the authors demonstrated that the drugs increased the levels of p21(Cip1/Waf1) and pro-apoptotic protein Bax and reduced the level of anti-apoptotic protein Bcl-2. Taken together, the results showed that the combination treatment of TCEE and AmB displays strong adjuvant effects, which are indicated by the inhibition of cell proliferation in 2 human cancer cell lines, RPMI7951 and MG63. These findings suggest possible therapeutic applications and alternative medicines using this drug combination.

  16. The role of lysosomes in BDE 47-mediated activation of mitochondrial apoptotic pathway in HepG2 cells.

    PubMed

    Liu, Xiaohui; Wang, Jian; Lu, Chengquan; Zhu, Chunyan; Qian, Bo; Li, Zhenwei; Liu, Chang; Shao, Jing; Yan, Jinsong

    2015-04-01

    Polybrominated diphenyl ethers (PBDEs) are a group of widely used flame retardants. The rising presence of PBDEs in human tissues has received considerable concerns with regard to potential health risks. While the mitochondrial-apoptotic pathway has been suggested in PBDEs-induced apoptosis, the role of lysosomes is yet to be understood. In the present study, HepG2 cells were exposed to BDE 47 at various concentrations and durations to establish the causal and temporal relationships among various cellular events, such as cell viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, and expression of cytochrome C and caspase 3. The involvement of lysosomes was simultaneously studied by evaluating lysosomal membrane permeability (LMP) and changes in the expression of cathepsin B, a lysosome hydrolase. In addition, a cathepsin B inhibitor (10 μM CA-074) was used to determine the involvement of lysosomes and potential interactions between lysosomes and mitochondria. Our results showed that ROS production was an initial response of HepG2 to BDE 47 exposure, followed by a decreased MMP; a loss of MMP caused additional ROS generation which acted to induce LMP; an increased LMP resulted in a release of cathepsin B which aggravated the loss of MMP leading to release of cytochrome C and caspase 3 and subsequent apoptosis. Pretreatment with CA-074 did not abolish the initial ROS generation, however, all downstream events were dramatically alleviated. Taken together, our data indicate that lysosomes might be involved in BDE 47-mediated mitochondrial-apoptotic pathway in HepG2 cells, possibly through feedback interactions between mitochondria and lysosomes.

  17. Cryptotanshinone induces melanoma cancer cells apoptosis via ROS-mitochondrial apoptotic pathway and impairs cell migration and invasion.

    PubMed

    Ye, Tinghong; Zhu, Shirui; Zhu, Yongxia; Feng, Qiang; He, Bing; Xiong, Yiong; Zhao, Lifeng; Zhang, Yiwen; Yu, Luoting; Yang, Li

    2016-08-01

    Melanoma is the most serious type of skin cancer because it is highly frequency of drug resistance and can spread earlier and more quickly than other skin cancers. The objective of this research was to investigate the anticancer effects of cryptotanshinone on human melanoma cells in vitro, and explored its mechanisms of action. Our results have shown that cryptotanshinone could inhibit cell proliferation in human melanoma cell lines A2058, A375, and A875 in a dose- and time-dependent manner. In addition, flow cytometry assay showed that cryptotanshinone inhibited the proliferation of human melanoma cell line A375 by blocking cell cycle progression in G2/M phase and inducing apoptosis in a concentration-dependent manner. Moreover, western blot analysis indicated that the occurrence of its apoptosis was associated with upregulation of cleaved caspases-3 and pro-apoptotic protein Bax while downregulation of anti-apoptotic protein Bcl-2. Meanwhile, cryptotanshinone could decrease the levels of reactive oxygen species (ROS). Furthermore, cryptotanshinone also blocked A375 cell migration and invasion in vitro which was associated with the downregulation with MMP-9. Taken together, these results suggested that cryptotanshinone might be a potential drug in human melanoma treatment by inhibiting proliferation, inducing apoptosis via ROS-mitochondrial apoptotic pathway and blocking cell migration and invasion.

  18. Colistin-induced apoptosis in PC12 cells: involvement of the mitochondrial apoptotic and death receptor pathways.

    PubMed

    Jiang, Hong; Li, Jichang; Zhou, Tiezhong; Wang, Chunhua; Zhang, Hua; Wang, Hongjun

    2014-05-01

    Colistin, a cyclic cationic polypeptide antibiotic that is used to treat infections, may cause neurotoxicity. However, whether colistin can induce apoptosis and the precise mechanism of apoptosis involved in PC12 cells remains to be determined. The aim of the present study was to determine reactive oxygen species (ROS) level and DNA damage, as well as apoptotic factors such as p53, cytochrome c, Bax, Bcl-2, Fas, Fas-L and caspase family via western blotting in PC12 cells treated with colistin sulfate. The results showed that colistin sulfate increased ROS levels significantly. An increase of ROS levels induces the release of cytochrome c and DNA damage. DNA damage can activate p53, which leads to the upregulation of Bax and downregulation of Bcl-2. The imbalance of Bax/Bcl-2 promotes additional release of cytochrome c. The release of cytochrome c contributes to the activation of caspase-9 and the subsequent activation of caspase-3. An increase of Fas and Fas-L induced the activation of caspase-8 leading to the activation of caspases-3, the latter induces apoptosis. Therefore, these results demonstrate that the apoptotic pathway of colistin-induced apoptosis in PC12 cells is involved in both the mitochondrial and death receptor pathway.

  19. The Key Role of Mitochondrial Apoptotic Pathway in the Cytotoxic Effect of Mushroom Extracts on Cancer Cells.

    PubMed

    Han, Mei; Ling, Ming-Tat; Chen, Jiezhong

    2015-01-01

    Mushroom extracts have been extensively studied for their medicinal effects. They can stimulate immune responses and thus have been explored in cancer treatment. Recently, it has also been shown that some mushroom extracts can produce direct cytotoxic effect on cancer cells. In this review, we summarize the cytotoxic effect of mushroom extracts in cancer treatment revealed by both in vitro and in vivo studies. We also summarize the current understanding of the mechanisms associated with such an effect with an emphasis on the mitochondrial apoptotic pathway. The recent finding that mushroom extracts have direct cytotoxic effects supplements their known immune stimulating effects. Thus, novel anticancer agents based on new findings from mushroom extracts may soon be added to the present pool of anticancer drugs. Specifically, we propose that nanodelivery of the bioactive compounds of mushroom extracts to mitochondria will further increase their potential treatment efficacy.

  20. Hydroxylation of multi-walled carbon nanotubes reduces their cytotoxicity by limiting the activation of mitochondrial mediated apoptotic pathway.

    PubMed

    Liu, Zhenbao; Liu, Yanfei; Peng, Dongming

    2014-04-01

    Hydroxylation of carbon nanotubes (CNTs) can enhance their dispersibility in water, and allows the capability to conjugate with other molecules for the expected applications. However, the cytotoxicity of hydroxylated CNTs has not been thoroughly investigated. Here, we compared the cytotoxicity of hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) on a human cell line with that of pristine multi-walled carbon nanotubes (p-MWCNTs). We showed that while both MWCNTs-OH and p-MWCNTs induced apoptosis in a time- and dose-dependent manner, MWCNTs-OH triggered a significantly milder cytotoxic response than that of p-MWCNTs. We further showed that such attenuated response could be attributed to a reduced disruption of the mitochondrial membrane potential (MMP), leading to the attenuation of both cytochrome c (cyt-c) release and activation of caspases. These findings suggest that MWCNTs-OH, could be more biocompatible for in vivo applications than that of p-MWCNTs by limiting the activation of the mitochondrial mediated apoptotic pathway.

  1. Carboxylation of multiwalled carbon nanotube enhanced its biocompatibility with L02 cells through decreased activation of mitochondrial apoptotic pathway.

    PubMed

    Liu, Zhenbao; Dong, Xia; Song, Liping; Zhang, Hailing; Liu, Lanxia; Zhu, Dunwan; Song, Cunxian; Leng, Xigang

    2014-03-01

    Modification of carbon nanotubes (CNTs) with carboxyl group is one of the widely used strategies to increase their water dispersibility. Various molecules can be further coupled to the surface of carboxylated CNTs for the desired applications. However, the effect of carboxylation of CNTs on their cytotoxicity is far from being completely understood. In this study, the impact of carboxylated multiwalled CNT (MWCNT-COOH) on human normal liver cell line L02 was studied and compared with pristine multiwalled CNT (p-MWCNT). The data accumulated in this study revealed that modification with carboxyl group reduced the toxicity of MWCNT on L02 cells, probably due to the decreased activation of mitochondria mediated apoptotic pathway. Both p-MWCNT and MWCNT-COOH, when reaching to certain concentration, induced significant decrease in the mitochondrial membrane potential, enhanced release of cytochrome c from the mitochondria to cytoplasm as well as activation of caspase-9, and -3. However, the changes induced by MWCNT-COOH were significantly milder than that by p-MWCNT. Our observation suggests that carboxylated MWCNTs might be safer for in vivo application as compared with p-MWCNT.

  2. Progesterone attenuates Aβ(25-35)-induced neuronal toxicity via JNK inactivation and progesterone receptor membrane component 1-dependent inhibition of mitochondrial apoptotic pathway.

    PubMed

    Qin, Yabin; Chen, Zesha; Han, Xiaolei; Wu, Honghai; Yu, Yang; Wu, Jie; Liu, Sha; Hou, Yanning

    2015-11-01

    Progesterone, which acts as a neurosteroid in nervous system, has been shown to have neuroprotective effects in different experiments in vitro and in vivo. Our previous study demonstrates that progesterone exerts neuroprotections in Alzheimer's disease-like rats. Present study attempted to evaluate the protective effects of progesterone on Aβ-treated neurons and potential mechanisms involved in neuroprotection. Results showed that treatment with progesterone protected primary cultured rat cortical neurons against Aβ(25-35)-induced apoptosis. Furthermore, we observed that progesterone alleviated mitochondrial dysfunction by rescuing mitochondrial membrane potential under Aβ challenge. Moreover, progesterone could also attenuate Bax/Bcl-2 proteins ratio upregulation and inhibit the activation of caspase-3 in Aβ-treated neurons. These indicate that progesterone attenuates Aβ(25-35)-induced neuronal toxicity by inhibiting mitochondria-associated apoptotic pathway. Both classic progesterone receptors (classic PR) and progesterone receptor membrane component 1 (PGRMC1), a special progesterone membrane receptor, are broadly expressed throughout the brain. The protective effect of progesterone was partially abolished by PGRMC1 inhibitor AG205 rather than classic PR antagonist RU486 in this study. Additionally, progesterone protected neurons by inhibiting Aβ-induced activation of JNK, which was an upstream signaling component in Aβ-induced mitochondria-associated apoptotic pathway. But this process was independent of PGRMC1. Taken together, these results suggest that progesterone exerts a protective effect against Aβ(25-35)-induced insults at least in part by two complementary pathways: (1) progesterone receptor membrane component 1-dependent inhibition of mitochondrial apoptotic pathway, and (2) blocking Aβ-induced JNK activation. The present study provides new insights into the mechanism by which progesterone brings neuroprotection. This article is part of a

  3. Pioglitazone alleviates the mitochondrial apoptotic pathway and mito-oxidative damage in the d-galactose-induced mouse model.

    PubMed

    Prakash, Atish; Kumar, Anil

    2013-09-01

    Chronic injection of d-galactose can cause gradual deterioration in learning and memory capacity, and activates oxidative stress, mitochondrial dysfunction and apoptotic cell death in the brain of mice. Thus, it serves as an animal model of ageing. Recent evidence has shown that mild cognitive impairment in humans might be alleviated by treatment with piogliatzone (peroxisome proliferator-activated receptor gamma (PPARγ) agonists). To continue exploring the effects of piogliatzone in this model, we focused on behavioural alteration, oxidative damage, mitochondrial dysfunction and apoptosis in d-galactose-induced mice. The ageing model was established by administration of d-galactose (100 mg/kg) for 6 weeks. Pioglitazone (10 and 30 mg/kg) and bisphenol A diglycidyl ether (15 mg/kg) were given daily to d-galactose-induced senescent mice. The cognitive behaviour of mice was monitored using the Morris water maze. The anti-oxidant status and apoptotic activity in the ageing mice was measured by determining mito-oxidative parameters and caspase-3 activity in brain tissue. Systemic administration of d-galactose significantly increased behavioural alterations, biochemical parameters, mitochondrial enzymes, and activations of caspase-3 and acetylcholinesterase enzyme activity as compared with the control group. Piogliatzone treatment significantly improved behavioural abnormalities, biochemical, cellular alterations, and attenuated the caspase-3 and acetylcholinesterase enzyme activity as compared with the control. Furthermore, pretreatment of BADGE (PPARγ antagonist) with pioglitazone reversed the protective effect of pioglitazone in d-galactose-induced mice. The present study highlights the protective effects of pioglitzone against d-galactose-induced memory dysfunction, mito-oxidative damage and apoptosis through activation of PPARγ receptors. These findings suggest that pioglitazone might be helpful for the prevention or alleviation of ageing.

  4. Mitochondrial apoptotic pathway activation in the atria of heart failure patients due to mitral and tricuspid regurgitation.

    PubMed

    Chang, Jen-Ping; Chen, Mien-Cheng; Liu, Wen-Hao; Lin, Yu-Sheng; Huang, Yao-Kuang; Pan, Kuo-Li; Ho, Wan-Chun; Fang, Chih-Yuan; Chen, Chien-Jen; Chen, Huang-Chung

    2015-08-01

    Apoptosis occurs in atrial cardiomyocytes in mitral and tricuspid valve disease. The purpose of this study was to examine the respective roles of the mitochondrial and tumor necrosis factor-α receptor associated death domain (TRADD)-mediated death receptor pathways for apoptosis in the atrial cardiomyocytes of heart failure patients due to severe mitral and moderate-to-severe tricuspid regurgitation. This study comprised eighteen patients (7 patients with persistent atrial fibrillation and 11 in sinus rhythm). Atrial appendage tissues were obtained during surgery. Three purchased normal human left atrial tissues served as normal controls. Moderately-to-severely myolytic cardiomyocytes comprised 59.7±22.1% of the cardiomyocytes in the right atria and 52.4±12.9% of the cardiomyocytes in the left atria of mitral and tricuspid regurgitation patients with atrial fibrillation group and comprised 58.4±24.8% of the cardiomyocytes in the right atria of mitral and tricuspid regurgitation patients with sinus rhythm. In contrast, no myolysis was observed in the normal human adult left atrial tissue samples. Immunohistochemical analysis showed expression of cleaved caspase-9, an effector of the mitochondrial pathways, in the majority of right atrial cardiomyocytes (87.3±10.0%) of mitral and tricuspid regurgitation patients with sinus rhythm, and right atrial cardiomyocytes (90.6±31.4%) and left atrial cardiomyocytes (70.7±22.0%) of mitral and tricuspid regurgitation patients with atrial fibrillation. In contrast, only 5.7% of cardiomyocytes of the normal left atrial tissues showed strongly positive expression of cleaved caspase-9. Of note, none of the atrial cardiomyocytes in right atrial tissue in sinus rhythm and in the fibrillating right and left atria of mitral and tricuspid regurgitation patients, and in the normal human adult left atrial tissue samples showed cleaved caspase-8 expression, which is a downstream effector of TRADD of the death receptor pathway

  5. IRF-1 transcriptionally upregulates PUMA, which mediates the mitochondrial apoptotic pathway in IRF-1-induced apoptosis in cancer cells.

    PubMed

    Gao, J; Senthil, M; Ren, B; Yan, J; Xing, Q; Yu, J; Zhang, L; Yim, J H

    2010-04-01

    Interferon regulatory factor-1 (IRF-1) is a transcription factor that acts as a tumor suppressor and causes apoptosis in cancer cells. We evaluated IRF-1-induced apoptosis in gastric cancer cell lines. We established stable clones in AGS cells that have a tetracycline-inducible IRF-1 expression system. We used these clones and recombinant adenovirus expressing IRF-1 to explore the mechanism of IRF-1-induced apoptosis in gastric cancer. Expression of IRF-1 causes apoptosis in gastric cancer cell lines as shown by phosphatidylserine exposure and cleavage of caspase-8, caspase-3, and Bid with the mitochondrial release of cytochrome c. However, inhibition of caspase-8 and Bid did not inhibit apoptosis and did not decrease cleaved caspase-9 or mitochondrial release of cytochrome c. We then show that IRF-1 upregulates PUMA (p53 upregulated modulator of apoptosis), which is known to activate apoptosis by the intrinsic pathway; this can be p53-independent. IRF-1 binds to distinct sites in the promoter of PUMA and activates PUMA transcription. Moreover, molecular markers of mitochondrial apoptosis are eliminated in PUMA knockout and knockdown cells and phosphatidylserine exposure is decreased dramatically. Finally, we show that IFN-gamma induces IRF-1-mediated upregulation of PUMA in cancer cells. We conclude that IRF-1 can induce apoptosis by the intrinsic pathway independent of the extrinsic pathway by upregulation of PUMA.

  6. Tetrahydrocurcumin reduces oxidative stress-induced apoptosis via the mitochondrial apoptotic pathway by modulating autophagy in rats after traumatic brain injury

    PubMed Central

    Gao, Yongyue; Zhuang, Zong; Gao, Shanting; Li, Xiang; Zhang, Zihuan; Ye, Zhennan; Li, Liwen; Tang, Chao; Zhou, Mengliang; Han, Xiao; Li, Jie

    2017-01-01

    Tetrahydrocurcumin (THC) has been identified as a multi-functional neuroprotective agent in numerous neurological disorders. Oxidative stress as a result of injury may induce neuronal apoptosis after traumatic brain injury (TBI). Treatment with THC may improve neurological function following TBI by attenuating oxidative stress and apoptosis and by enhancing autophagy. The purpose of this study was to investigate the mechanism of neuroprotection by THC against oxidative stress-induced neuronal apoptosis after TBI. We hypothesized that neuroprotection by THC may involve modulation of autophagy and the mitochondria apoptotic pathway. We used western blot analysis to evaluate the effect of THC on proteins involved in mitochondrial autophagy and apoptosis after TBI. The terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and immunofluorescence staining were used to confirm the role of THC in apoptosis and autophagy, respectively. THC-induced neuroprotection was assessed by neurological severity scoring (NSS) and by measuring the brain water content. We demonstrated that treatment with THC increased expression of autophagy-associated proteins LC3-II and Beclin-1 at 24 h post-TBI. Treatment with THC also reduced expression of malondialdehyde (MDA) and increased glutathione peroxidase (GPx) activity. Further, treatment with THC attenuated apoptosis by modulating mitochondrial apoptosis and reducing oxidative stress. Treatment with 3-methyladenine (3-MA) mitigated autophagy activation and reversed the inhibitory effect of THC on the translocation of Bax to the mitochondrial membrane. Moreover, treatment with THC improved neurological function and reduced the brain water content in rats after TBI. We concluded that the neuroprotective effects of THC are mediated by enhancing autophagy activation and by attenuation of oxidative stress and apoptosis after TBI, probably by modulating the mitochondrial apoptotic pathway. We suggest that THC may be an

  7. Phenolic extract from oleaster (Olea europaea var. Sylvestris) leaves reduces colon cancer growth and induces caspase-dependent apoptosis in colon cancer cells via the mitochondrial apoptotic pathway

    PubMed Central

    Belarbi, Meriem; Dumont, Adélie; de Rosny, Charlotte; Aboura, Ikram; Ghanemi, Fatima Zahra; Murtaza, Babar; Patoli, Danish; Thomas, Charles; Apetoh, Lionel; Rébé, Cédric; Delmas, Dominique; Akhtar Khan, Naim; Ghiringhelli, François; Rialland, Mickael; Hichami, Aziz

    2017-01-01

    Dietary polyphenols, derived from natural products, have received a great interest for their chemopreventive properties against cancer. In this study, we investigated the effects of phenolic extract of the oleaster leaves (PEOL) on tumor growth in mouse model and on cell death in colon cancer cell lines. We assessed the effect of oleaster leaf infusion on HCT116 (human colon cancer cell line) xenograft growth in athymic nude mice. We observed that oleaster leaf polyphenol-rich infusion limited HCT116 tumor growth in vivo. Investigations of PEOL on two human CRC cell lines showed that PEOL induced apoptosis in HCT116 and HCT8 cells. We demonstrated an activation of caspase-3, -7 and -9 by PEOL and that pre-treatment with the pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), prevented PEOL-induced cell death. We observed an involvement of the mitochondrial pathway in PEOL-induced apoptosis evidenced by reactive oxygen species (ROS) production, a decrease of mitochondrial membrane potential, and cytochrome c release. Increase in intracellular Ca2+ concentration induced by PEOL represents the early event involved in mitochondrial dysfunction, ROS-induced endoplasmic reticulum (ER) stress and apoptosis induced by PEOL, as ruthenium red, an inhibitor of mitochondrial calcium uptake inhibited apoptotic effect of PEOL, BAPTA/AM inhibited PEOL-induced ROS generation and finally, N-acetyl-L-cysteine reversed ER stress and apoptotic effect of PEOL. These results demonstrate that polyphenols from oleaster leaves might have a strong potential as chemopreventive agent in colorectal cancer. PMID:28212423

  8. Phenolic extract from oleaster (Olea europaea var. Sylvestris) leaves reduces colon cancer growth and induces caspase-dependent apoptosis in colon cancer cells via the mitochondrial apoptotic pathway.

    PubMed

    Zeriouh, Wafa; Nani, Abdelhafid; Belarbi, Meriem; Dumont, Adélie; de Rosny, Charlotte; Aboura, Ikram; Ghanemi, Fatima Zahra; Murtaza, Babar; Patoli, Danish; Thomas, Charles; Apetoh, Lionel; Rébé, Cédric; Delmas, Dominique; Akhtar Khan, Naim; Ghiringhelli, François; Rialland, Mickael; Hichami, Aziz

    2017-01-01

    Dietary polyphenols, derived from natural products, have received a great interest for their chemopreventive properties against cancer. In this study, we investigated the effects of phenolic extract of the oleaster leaves (PEOL) on tumor growth in mouse model and on cell death in colon cancer cell lines. We assessed the effect of oleaster leaf infusion on HCT116 (human colon cancer cell line) xenograft growth in athymic nude mice. We observed that oleaster leaf polyphenol-rich infusion limited HCT116 tumor growth in vivo. Investigations of PEOL on two human CRC cell lines showed that PEOL induced apoptosis in HCT116 and HCT8 cells. We demonstrated an activation of caspase-3, -7 and -9 by PEOL and that pre-treatment with the pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), prevented PEOL-induced cell death. We observed an involvement of the mitochondrial pathway in PEOL-induced apoptosis evidenced by reactive oxygen species (ROS) production, a decrease of mitochondrial membrane potential, and cytochrome c release. Increase in intracellular Ca2+ concentration induced by PEOL represents the early event involved in mitochondrial dysfunction, ROS-induced endoplasmic reticulum (ER) stress and apoptosis induced by PEOL, as ruthenium red, an inhibitor of mitochondrial calcium uptake inhibited apoptotic effect of PEOL, BAPTA/AM inhibited PEOL-induced ROS generation and finally, N-acetyl-L-cysteine reversed ER stress and apoptotic effect of PEOL. These results demonstrate that polyphenols from oleaster leaves might have a strong potential as chemopreventive agent in colorectal cancer.

  9. Triggering Apoptotic Death of Human Malignant Melanoma A375.S2 Cells by Bufalin: Involvement of Caspase Cascade-Dependent and Independent Mitochondrial Signaling Pathways

    PubMed Central

    Hsiao, Yu-Ping; Yu, Chun-Shu; Yu, Chien-Chih; Yang, Jai-Sing; Chiang, Jo-Hua; Lu, Chi-Cheng; Huang, Hui-Ying; Tang, Nou-Ying; Yang, Jen-Hung; Huang, An-Cheng; Chung, Jing-Gung

    2012-01-01

    Bufalin was obtained from the skin and parotid venom glands of toad and has been shown to induce cytotoxic effects in various types of cancer cell lines, but there is no report to show that whether bufalin affects human skin cancer cells. The aim of this investigation was to study the effects of bufalin on human malignant melanoma A375.S2 cells and to elucidate possible mechanisms involved in induction of apoptosis. A375.S2 cells were treated with different concentrations of bufalin for a specific time period and investigated for effects on apoptotic analyses. Our results indicated that cells after exposure to bufalin significantly decreased cell viability, and induced cell morphological changes and chromatin condensation in a concentration-dependent manner. Flow cytometric assays indicated that bufalin promoted ROS productions, loss of mitochondrial membrane potential (ΔΨm), intracellular Ca2+ release, and nitric oxide (NO) formations in A375.S2 cells. Additionally, the apoptotic induction of bufalin on A375.S2 cells resulted from mitochondrial dysfunction-related responses (disruption of the ΔΨm and releases of cytochrome c, AIF, and Endo G), and activations of caspase-3, caspase-8 and caspase-9 expressions. Based on those observations, we suggest that bufalin-triggered apoptosis in A375.S2 cells is correlated with extrinsic- and mitochondria-mediated multiple signal pathways. PMID:22719785

  10. The p53-inducible gene 3 involved in flavonoid-induced cytotoxicity through the reactive oxygen species-mediated mitochondrial apoptotic pathway in human hepatoma cells.

    PubMed

    Zhang, Qiang; Cheng, Guangdong; Qiu, Hongbin; Zhu, Liling; Ren, Zhongjuan; Zhao, Wei; Zhang, Tao; Liu, Lei

    2015-05-01

    Flavonoids have been reported to exhibit prooxidant cytotoxicity against cancer cells, but the underlying mechanism is still poorly understood. Here we investigated the potential mechanism that p53-inducible gene 3 (PIG3), a NADPH:quinone oxidoreductase, mediated the prooxidant cytotoxicity of flavonoids on human hepatoma HepG2 cells. The results showed that flavonoids (apigenin, luteolin, kaempferol, and quercetin) inhibited the growth of HepG2 cells in a dosage- and time-dependent manner, and induced the morphological changes characteristic of apoptosis in HepG2 cells. We also found that expression of PIG3 was increased markedly in HepG2 cells treated with flavonoids at both mRNA and protein levels, which was accompanied by increased intracellular ROS production and a decreased mitochondrial membrane potential (ΔΨm). All these effects were largely reversed through knockdown of the PIG3 gene in HepG2 cells. Western blotting indicated that flavonoids increased cytochrome c release, upregulated the ratio of Bax/Bcl-2, and activated the caspases-9 and -3. Moreover, knockdown of PIG3 could reverse the changes of these apoptotic-related proteins. These results suggest that PIG3 plays an important role in regulating the prooxidant activity and apoptosis-inducing action of flavonoids on HepG2 cells though the ROS-triggered mitochondrial apoptotic pathway.

  11. Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways.

    PubMed

    Chong, Zhao Zhong; Shang, Yan Chen; Hou, Jinling; Maiese, Kenneth

    2010-01-01

    Although essential for the development of the nervous system, Wnt1 also has been associated with neurodegenerative disease and cognitive loss during periods of oxidative stress. Here we show that endogenous expression of Wnt1 is suppressed during oxidative stress in both in vitro and in vivo experimental models. Loss of endogenous Wnt1 signaling directly correlates with neuronal demise and increased functional deficit, illustrating that endogenous neuronal Wnt1 offers a vital level of intrinsic cellular protection against oxidative stress. Furthermore, transient overexpression of Wnt1 or application of exogenous Wnt1 recombinant protein is necessary to preserve neurological function and rescue neurons from apoptotic membrane phosphatidylserine externalization and genomic DNA degradation, since blockade of Wnt1 signaling with a Wnt1 antibody or dickkopf related protein 1 abrogates neuronal protection by Wnt1. Wnt1 ultimately relies upon the activation of Akt1, the modulation of mitochondrial membrane permeability, and the release of cytochrome c to control the apoptotic cascade, since inhibition of Wnt1 signaling, the phosphatidylinositol 3-kinase pathway, or Akt1 activity abrogates the ability of Wnt1 to block these apoptotic components. Our work identifies Wnt1 and its downstream signaling as cellular targets with high clinical potential for novel treatment strategies for multiple disorders precipitated by oxidative stress.

  12. Bisdemethoxycurcumin exerts pro-apoptotic effects in human pancreatic adenocarcinoma cells through mitochondrial dysfunction and a GRP78-dependent pathway

    PubMed Central

    Yang, Haopeng; Fan, Shengjun; An, Yu; Wang, Xin; Pan, Yan; Xiaokaiti, Yilixiati; Duan, Jianhui; Li, Xin; Tie, Lu; Ye, Min; Li, Xuejun

    2016-01-01

    Pancreatic cancer is a highly aggressive malignancy, which is intrinsically resistant to current chemotherapies. Herein, we investigate whether bisdemethoxycurcumin (BDMC), a derivative of curcumin, potentiates gemcitabine in human pancreatic cancer cells. The result suggests that BDMC sensitizes gemcitabine by inducing mitochondrial dysfunctions and apoptosis in PANC-1 and MiaPaCa-2 pancreatic cancer cells. Utilizing two-dimensional gel electrophoresis and mass spectrometry, we identify 13 essential proteins with significantly altered expressions in response to gemcitabine alone or combined with BDMC. Protein-protein interaction network analysis pinpoints glucose-regulated protein 78 (GRP78) as the key hub activated by BDMC. We then reveal that BDMC upregulates GRP78 and facilitates apoptosis through eIF2α/CHOP pathway. Moreover, DJ-1 and prohibitin, two identified markers of chemoresistance, are increased by gemcitabine in PANC-1 cells. This could be meaningfully reversed by BDMC, suggesting that BDMC partially offsets the chemoresistance induced by gemcitabine. In summary, these findings show that BDMC promotes apoptosis through a GRP78-dependent pathway and mitochondrial dysfunctions, and potentiates the antitumor effect of gemcitabine in human pancreatic cancer cells. PMID:27845899

  13. Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

    SciTech Connect

    Ji Lili; Chen Ying; Liu Tianyu; Wang Zhengtao

    2008-09-15

    Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 {mu}M)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 {mu}M clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 {mu}M) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.

  14. Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

    2010-03-01

    The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

  15. Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways

    SciTech Connect

    Chang, Chun-Yu; Shen, Chao-Yu; Kang, Chao-Kai; Sher, Yuh-Pyng; Sheu, Wayne H.-H.; Chang, Chia-Che; Lee, Tsung-Han

    2014-09-15

    Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100 μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways. - Highlights: • Oxidized LDL induced cytotoxicity and apoptosis in HK-2 cells. • Pretreatment with taurine attenuated oxLDL-induced nephrotoxicity. • Taurine protected against renal damages through inhibition of ROS generation. • Taurine prevented apoptosis through modulation of the p53 phosphorylation.

  16. CD147 interacts with NDUFS6 in regulating mitochondrial complex I activity and the mitochondrial apoptotic pathway in human malignant melanoma cells.

    PubMed

    Luo, Z; Zeng, W; Tang, W; Long, T; Zhang, J; Xie, X; Kuang, Y; Chen, M; Su, J; Chen, X

    2014-01-01

    Malignant melanoma (MM) is one of the most lethal tumors and is characterized by high invasiveness, frequent metastasis, and resistance to chemotherapy. The risk of metastatic MM is accompanied by disordered energy metabolism involving the oxidative phosphorylation (OXPHOS) process, which is largely carried out in mitochondrial complexes. Complex I is the first and largest mitochondrial enzyme complex associated with this process. CD147 is a transmembrane glycoprotein mainly expressed on the cell surface, and also appears in the cytoplasm in some tumors. We found that CD147 is often translocated to the cytoplasm in metastatic MM specimens as compared to primary MM. We also demonstrated high expression of CD147 in isolated mitochondrial fractions of A375 cells. The yeast two-hybrid (Y2H) assay identified NDUFS6 (which encodes a subunit of mitochondrial respiratory chain complex I) as a candidate that interacts with CD147 and depletion of CD147 in A375 cells significantly decreased complex I enzyme activity. We also showed that CD147 increased the viability of A375 cells exposed to berberine-induced mitochondrial damage, and protected them from apoptosis through a mitochondrial-dependent pathway. This finding was confirmed by adding exogenous Bcl-2 to A375 cell cultures. In summary, our results identify the existence of CD147 in human melanoma cell mitochondria. They indicate that CD147 appears to regulate complex I activity and apoptosis in MM by interacting with mitochondrial NDUFS6. Our findings provide new insight into the function of CD147 and identify it as a promising therapeutic target in melanoma through disruption of the energy metabolism.

  17. The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells.

    PubMed

    Hsin, Yi-Hong; Chen, Chun-Feng; Huang, Shing; Shih, Tung-Sheng; Lai, Ping-Shan; Chueh, Pin Ju

    2008-07-10

    Nanomaterials and nanoparticles have received considerable attention recently because of their unique properties and diverse biotechnology and life sciences applications. Nanosilver products, which have well-known antimicrobial properties, have been used extensively in a range of medical settings. Despite the widespread use of nanosilver products, relatively few studies have been undertaken to determine the biological effects of nanosilver exposure. The purpose of this study was to evaluate the toxicity of nanosilver and to elucidate possible molecular mechanisms underlying the biological effects of nanosilver. Here, we show that nanosilver is cytotoxic, inducing apoptosis in NIH3T3 fibroblast cells. Treatment with nanosilver induced the release of cytochrome c into the cytosol and translocation of Bax to mitochondria, indicating that nanosilver-mediated apoptosis is mitochondria-dependent. Nanosilver-induced apoptosis was associated with the generation of reactive oxygen species (ROS) and JNK activation, and inhibition of either ROS or JNK attenuated nanosilver-induced apoptosis. In nanosilver-resistant HCT116 cells, up-regulation of the anti-apoptotic proteins, Bcl-2 appeared to be associated with a diminished apoptotic response. Taken together, our results provide the first evidence for a molecular mechanism of nanosilver cytotoxicity, showing that nanosilver acts through ROS and JNK to induce apoptosis via the mitochondrial pathway.

  18. A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

    PubMed Central

    2011-01-01

    Background Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd2 [S(-)C2, N-dmpa]2 (μ-dppe)Cl2} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. Methods B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. Results Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. Conclusions The

  19. CQ synergistically sensitizes human colorectal cancer cells to SN-38/CPT-11 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk.

    PubMed

    Chen, Pinjia; Luo, Xiaoyong; Nie, Peipei; Wu, Baoyan; Xu, Wei; Shi, Xinpeng; Chang, Haocai; Li, Bing; Yu, Xiurong; Zou, Zhengzhi

    2017-03-01

    Autophagy plays a key role in supporting cell survival against chemotherapy-induced apoptosis. In this study, we found the chemotherapy agent SN-38 induced autophagy in colorectal cancer (CRC) cells. However, inhibition of autophagy using a small molecular inhibitor 3-methyladenine (3-MA) and ATG5 siRNA did not increase SN-38-induced cytotoxicity in CRC cells. Notably, another autophagy inhibitor chloroquine (CQ) synergistically enhanced the anti-tumor activity of SN-38 in CRC cells with wild type (WT) p53. Subsequently, we identified a potential mechanism of this cooperative interaction by showing that CQ and SN-38 acted together to trigger reactive oxygen species (ROS) burst, upregulate p53 expression, elicit the loss of lysosomal membrane potential (LMP) and mitochondrial membrane potential (∆ψm). In addition, ROS induced by CQ plus SN-38 upregulated p53 levels by activating p38, conversely, p53 stimulated ROS. These results suggested that ROS and p53 reciprocally promoted each other's production and cooperated to induce CRC cell death. Moreover, we showed induction of ROS and p53 by the two agents provoked the loss of LMP and ∆ψm. Altogether, all results suggested that CQ synergistically sensitized human CRC cells with WT p53 to SN-38 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk. Lastly, we showed that CQ could enhance CRC cells response to CPT-11 (a prodrug of SN-38) in xenograft models. Thus the combined treatment might represent an attractive therapeutic strategy for the treatment of CRC.

  20. HIF-1α inhibition by 2-methoxyestradiol induces cell death via activation of the mitochondrial apoptotic pathway in acute myeloid leukemia.

    PubMed

    Zhe, Nana; Chen, Shuya; Zhou, Zhen; Liu, Ping; Lin, Xiaojing; Yu, Meisheng; Cheng, Bingqing; Zhang, Yaming; Wang, Jishi

    2016-06-02

    The bone marrow microenvironment plays an important role in the development and progression of AML. Leukemia stem cells are in a hypoxic condition, which induces the expression of HIF-1α. Aberrant activation of HIF-1α is implicated in the poor prognosis of patients with acute myeloid leukemia (AML). Herein, we investigated the expression of HIF-1α in AML and tested 2-methoxyestradiol (2ME2) as a candidate HIF-1α inhibitor for the treatment of AML. We found that HIF-1α was overexpressed in AML. HIF-1α suppression by 2ME2 significantly induced apoptosis of AML cells, and it outperformed traditional chemotherapy drugs such as cytarabine. At the same time, 2ME2 downregulated the transcriptional levels of VEGF, GLUT1 and HO-1 in cellular assays. Additionally, 2ME2 displayed antileukemia activity in bone marrow blasts from AML patients, but showed little effect on normal cells. 2ME2-induced activation of mitochondrial apoptotic pathway is mediated by reactive oxygen species (ROS), which decreased the slight effect of drug on normal cells. Our data show that supression of HIF-1α expression significantly reduced the survival of AML cell lines, suggesting that 2ME2 may represent a powerful therapeutic approach for patients with AML.

  1. The mitochondrial apoptotic pathway is induced by Cu(II) antineoplastic compounds (Casiopeínas(®)) in SK-N-SH neuroblastoma cells after short exposure times.

    PubMed

    García-Ramos, Juan Carlos; Gutiérrez, Anllely Grizett; Vázquez-Aguirre, Adriana; Toledano-Magaña, Yanis; Alonso-Sáenz, Ana Luisa; Gómez-Vidales, Virginia; Flores-Alamo, Marcos; Mejía, Carmen; Ruiz-Azuara, Lena

    2017-02-01

    The family of Copper(II) coordination compounds Casiopeínas(®) (Cas) has shown antiproliferative activity in several tumour lines by oxidative cellular damage and mitochondrial dysfunction that lead to cell death through apoptotic pathways. The goal of this work is looking for the functional mechanism of CasIIgly, CasIIIia and CasIIIEa in neuroblastoma metastatic cell line SK-N-SH, a paediatric extra-cranial tumour which is refractory to several anti-carcinogenic agents. All Cas have shown higher antiproliferative activity than cisplatin (IC50 = 123 μM) with IC50 values of 18, 22 and 63 µM for CasIIgly, CasIIIEa and CasIIIia, respectively. At low concentrations and early times (4 h), these compounds cause a disruption of the mitochondrial transmembrane potential (Δψm). Concomitantly, an important depletion of intracellular glutathione and an increase of reactive oxygen species (ROS) hydrogen peroxide and radical superoxide were observed. On the other side, the lower cytotoxic effect of Casiopeínas on cultures of human peripheral blood lymphocytes (IC50CasIIgly  = 1720 µM, IC50 CasIIIEa  = 3860 µM and IC50 CasIIIia  = 4700 µM) show the selectivity of these compounds over the tumour cells compared with the non-transformed cells. Chemically, glutathione (GSH) interacts with Casiopeínas(®) through the coordination of sulphur atom to the metal centre, process which facilitates the electron transfer to get Cu(I), GSSG and the posterior production of ROS. Additionally, the molecular structure of CasIIIia as nitrate is reported. These results have shown that the anticarcinogenic activity of Casiopeínas(®) on neuroblastoma SK-N-SH is through mitochondrial apoptosis due to the enhanced pro-oxidant environment promoted by the presence of the coordination copper compounds.

  2. PUMA-mediated mitochondrial apoptotic disruption by hypoxic postconditioning.

    PubMed

    Li, YuZhen; Guo, Qi; Liu, XiuHua; Wang, Chen; Song, DanDan

    2015-08-01

    Postconditioning can reduce ischemia-reperfusion (I/R)-induced cardiomyocyte apoptosis by targeting mitochondria. p53 upregulated modulator of apoptosis (PUMA) is involved in lethal I/R injury. Here, we hypothesized that postconditioning might inhibit mitochondrial pathway-mediated cardiomyocyte apoptosis by controlling PUMA expression. The cultured neonatal rat cardiomyocytes underwent 3 h of hypoxia and 3 h of reoxygenation. Postconditioning consisted of three cycles of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Hypoxic postconditioning reduced the levels of PUMA mRNA and protein. Concomitantly, the loss of mitochondrial membrane potential, cytochrome c release and caspase-3 activation were decreased significantly by postconditioning. Overexpression of PUMA increased greatly not only the number of apoptotic cardiomyocytes, but also the collapse of mitochondrial membrane potential, cytochrome c release and caspase-3 activation under postconditioning condition. The data suggest that reduction of PUMA expression mediates the endogenous cardioprotective mechanisms of postconditioning by disrupting mitochondrial apoptotic pathway.

  3. Zinc ferrite nanoparticles activate IL-1b, NFKB1, CCL21 and NOS2 signaling to induce mitochondrial dependent intrinsic apoptotic pathway in WISH cells

    SciTech Connect

    Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Ahmad, Javed; Siddiqui, Maqsood A.; Dwivedi, Sourabh; Khan, Shams T.; Musarrat, Javed

    2013-12-01

    localization of NPs. • ZnFe{sub 2}O{sub 4}-NPs induce DNA damage and mitochondrial dysfunction in WISH cells. • ZnFe{sub 2}O{sub 4}-NPs activate inflammatory and oxidative stress signaling in WISH cells. • Elevation of p53, CASP 3, bax and bcl 2 genes affirms intrinsic apoptotic pathway.

  4. Human chorionic gonadotropin suppresses human breast cancer cell growth directly via p53-mediated mitochondrial apoptotic pathway and indirectly via ovarian steroid secretion.

    PubMed

    Yuri, Takashi; Kinoshita, Yuichi; Emoto, Yuko; Yoshizawa, Katsuhiko; Tsubura, Airo

    2014-03-01

    The tumor-suppressive effects of human chorionic gonadotropin (hCG) against human breast cancer cells were examined. In cell viability assays, hCG inhibited the growth of three human breast cancer cell lines (estrogen receptor (ER)-positive KPL-1 and MCF-7, and ER-negative MKL-F cells), and the growth inhibition activity of hCG was most pronounced against KPL-1 cells (luteinizing hormone/chorionic gonadotropin receptor (LHCGR)-positive and luminal-A subtype). In hCG-treated KPL-1 cells, immunoblotting analysis revealed the expression of tumor suppressor protein p53 peaking at 12 h following treatment, followed by cleavage of caspase-9 and caspase-3 at 24 h and 48 h, respectively. KPL-1-transplanted athymic mice were divided into 3 groups: a sham-treated group that received an inoculation of KPL-1 cells at 6 weeks of age followed by daily intraperitoneal (i.p.) injection of saline; an in vitro hCG-treated KPL-1 group that received an inoculation of KPL-1 cells pre-treated with 100 IU/ml hCG in vitro for 48 h at 6 weeks of age, followed by daily i.p. injection of saline; and an in vivo hCG-treated group that received an KPL-1 cell inoculation at 6 weeks of age, followed by daily i.p. injection of 100 IU hCG. The daily injections of saline or hCG continued until the end of the experiment when mice reached 11 weeks of age. KPL-1 tumor growth was retarded in in vitro and in vivo hCG-treated mice compared to sham-treated controls, and the final tumor volume and tumor weight tended to be suppressed in the in vitro hCG-treated group and were significantly suppressed in the in vivo hCG-treated group. In vivo 100-IU hCG injections for 5 weeks elevated serum estradiol levels (35.7 vs. 23.5 pg/ml); thus, the mechanisms of hCG action may be directly coordinated via the p53-mediated mitochondrial apoptotic pathway and indirectly through ovarian steroid secretion that elevates estrogen levels. It is thus concluded that hCG may be an attractive agent for treating human breast

  5. ER-Dependent Ca++-mediated Cytosolic ROS as an Effector for Induction of Mitochondrial Apoptotic and ATM-JNK Signal Pathways in Gallic Acid-treated Human Oral Cancer Cells.

    PubMed

    Lu, Yao-Cheng; Lin, Meng-Liang; Su, Hong-Lin; Chen, Shih-Shun

    2016-02-01

    Release of calcium (Ca(++)) from the endoplasmic reticulum (ER) has been proposed to be involved in induction of apoptosis by oxidative stress. Using inhibitor of ER Ca(++) release dantrolene and inhibitor of mitochondrial Ca(++) uptake Ru-360, we demonstrated that Ca(++) release from the ER was associated with generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, and apoptosis of human oral cancer (OC) cells induced by gallic acid (GA). Small interfering RNA-mediated suppression of protein kinase RNA-like endoplasmic reticulum kinase inhibited tunicamycin-induced induction of 78 kDa glucose-regulated protein, C/EBP homologous protein, pro-caspase-12 cleavage, cytosolic Ca(++) increase and apoptosis, but did not attenuate the increase in cytosolic Ca(++) level and apoptosis induced by GA. Ataxia telangiectasia mutated (ATM)-mediated c-Jun N-terminal kinase (JNK) phosphorylation and apoptosis by GA was blocked by dantrolene. The specificity of ROS-mediated ATM-JNK activation was confirmed by treatment with N-acetylcysteine, a ROS scavenger. Blockade of ATM activation by specific inhibitor KU55933, short hairpin RNA, or kinase-dead ATM overexpression suppressed JNK phosphorylation but did not completely inhibit cytosolic ROS production, mitochondrial cytochrome c release, pro-caspase-3 cleavage, and apoptosis induced by GA. Taken together, these results indicate that GA induces OC cell apoptosis by inducing the activation of mitochondrial apoptotic and ATM-JNK signal pathways, likely through ER Ca(++)-mediated ROS production.

  6. Mangiferin Attenuates Diabetic Nephropathy by Inhibiting Oxidative Stress Mediated Signaling Cascade, TNFα Related and Mitochondrial Dependent Apoptotic Pathways in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Pal, Pabitra Bikash; Sinha, Krishnendu; Sil, Parames C.

    2014-01-01

    Oxidative stress plays a crucial role in the progression of diabetic nephropathy in hyperglycemic conditions. It has already been reported that mangiferin, a natural C-glucosyl xanthone and polyhydroxy polyphenol compound protects kidneys from diabetic nephropathy. However, little is known about the mechanism of its beneficial action in this pathophysiology. The present study, therefore, examines the detailed mechanism of the beneficial action of mangiferin on STZ-induced diabetic nephropathy in Wister rats as the working model. A significant increase in plasma glucose level, kidney to body weight ratio, glomerular hypertrophy and hydropic changes as well as enhanced nephrotoxicity related markers (BUN, plasma creatinine, uric acid and urinary albumin) were observed in the experimental animals. Furthermore, increased oxidative stress related parameters, increased ROS production and decreased the intracellular antioxidant defenses were detected in the kidney. Studies on the oxidative stress mediated signaling cascades in diabetic nephropathy demonstrated that PKC isoforms (PKCα, PKCβ and PKCε), MAPKs (p38, JNK and ERK1/2), transcription factor (NF-κB) and TGF-β1 pathways were involved in this pathophysiology. Besides, TNFα was released in this hyperglycemic condition, which in turn activated caspase 8, cleaved Bid to tBid and finally the mitochorndia-dependent apoptotic pathway. In addition, oxidative stress also disturbed the proapoptotic-antiapoptotic (Bax and Bcl-2) balance and activated mitochorndia-dependent apoptosis via caspase 9, caspase 3 and PARP cleavage. Mangiferin treatment, post to hyperglycemia, successfully inhibited all of these changes and protected the cells from apoptotic death. PMID:25233093

  7. Apoptotic effect of novel Schiff Based CdCl2(C14H21N3O2) complex is mediated via activation of the mitochondrial pathway in colon cancer cells

    PubMed Central

    Hajrezaie, Maryam; Paydar, Mohammadjavad; Looi, Chung Yeng; Moghadamtousi, Soheil Zorofchian; Hassandarvish, Pouya; Salga, Muhammad Saleh; Karimian, Hamed; Shams, Keivan; Zahedifard, Maryam; Majid, Nazia Abdul; Ali, Hapipah Mohd; Abdulla, Mahmood Ameen

    2015-01-01

    The development of metal-based agents has had a tremendous role in the present progress in cancer chemotherapy. One well-known example of metal-based agents is Schiff based metal complexes, which hold great promise for cancer therapy. Based on the potential of Schiff based complexes for the induction of apoptosis, this study aimed to examine the cytotoxic and apoptotic activity of a CdCl2(C14H21N3O2) complex on HT-29 cells. The complex exerted a potent suppressive effect on HT-29 cells with an IC50 value of 2.57 ± 0.39 after 72 h of treatment. The collapse of the mitochondrial membrane potential and the elevated release of cytochrome c from the mitochondria to the cytosol indicate the involvement of the intrinsic pathway in the induction of apoptosis. The role of the mitochondria-dependent apoptotic pathway was further proved by the significant activation of the initiator caspase-9 and the executioner caspases-3 and -7. In addition, the activation of caspase-8, which is associated with the suppression of NF-κB translocation to the nucleus, also revealed the involvement of the extrinsic pathway in the induced apoptosis. The results suggest that the CdCl2(C14H21N3O2) complex is able to induce the apoptosis of colon cancer cells and is a potential candidate for future cancer studies. PMID:25764970

  8. Esculetin, a natural coumarin compound, evokes Ca(2+) movement and activation of Ca(2+)-associated mitochondrial apoptotic pathways that involved cell cycle arrest in ZR-75-1 human breast cancer cells.

    PubMed

    Chang, Hong-Tai; Chou, Chiang-Ting; Lin, You-Sheng; Shieh, Pochuen; Kuo, Daih-Huang; Jan, Chung-Ren; Liang, Wei-Zhe

    2016-04-01

    Esculetin (6,7-dihydroxycoumarin), a derivative of coumarin compound, is found in traditional medicinal herbs. It has been shown that esculetin triggers diverse cellular signal transduction pathways leading to regulation of physiology in different models. However, whether esculetin affects Ca(2+) homeostasis in breast cancer cells has not been explored. This study examined the underlying mechanism of cytotoxicity induced by esculetin and established the relationship between Ca(2+) signaling and cytotoxicity in human breast cancer cells. The results showed that esculetin induced concentration-dependent rises in the intracellular Ca(2+) concentration ([Ca(2+)]i) in ZR-75-1 (but not in MCF-7 and MDA-MB-231) human breast cancer cells. In ZR-75-1 cells, this Ca(2+) signal response was reduced by removing extracellular Ca(2+) and was inhibited by the store-operated Ca(2+) channel blocker 2-aminoethoxydiphenyl borate (2-APB). In Ca(2+)-free medium, pre-treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished esculetin-induced [Ca(2+)]i rises. Conversely, incubation with esculetin abolished TG-induced [Ca(2+)]i rises. Esculetin induced cytotoxicity that involved apoptosis, as supported by the reduction of mitochondrial membrane potential and the release of cytochrome c and the proteolytic activation of caspase-9/caspase-3, which were partially reversed by pre-chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Moreover, esculetin increased the percentage of cells in G2/M phase and regulated the expressions of p53, p21, CDK1, and cyclin B1. Together, in ZR-75-1 cells, esculetin induced [Ca(2+)]i rises by releasing Ca(2+) from the ER and causing Ca(2+) influx through 2-APB-sensitive store-operated Ca(2+) entry. Furthermore, esculetin activated Ca(2+)-associated mitochondrial apoptotic pathways that involved G2/M cell cycle arrest. Graphical abstract The summary of esculetin

  9. Computational modeling of apoptotic signaling pathways induced by cisplatin

    PubMed Central

    2012-01-01

    Background Apoptosis is an essential property of all higher organisms that involves extremely complex signaling pathways. Mathematical modeling provides a rigorous integrative approach for analyzing and understanding such intricate biological systems. Results Here, we constructed a large-scale, literature-based model of apoptosis pathways responding to an external stimulus, cisplatin. Our model includes the key elements of three apoptotic pathways induced by cisplatin: death receptor-mediated, mitochondrial, and endoplasmic reticulum-stress pathways. We showed that cisplatin-induced apoptosis had dose- and time-dependent characteristics, and the level of apoptosis was saturated at higher concentrations of cisplatin. Simulated results demonstrated that the effect of the mitochondrial pathway on apoptosis was the strongest of the three pathways. The cross-talk effect among pathways accounted for approximately 25% of the total apoptosis level. Conclusions Using this model, we revealed a novel mechanism by which cisplatin induces dose-dependent cell death. Our finding that the level of apoptosis was affected by not only cisplatin concentration, but also by cross talk among pathways provides in silico evidence for a functional impact of system-level characteristics of signaling pathways on apoptosis. PMID:22967854

  10. Cancer therapeutics: Targeting the apoptotic pathway.

    PubMed

    Khan, Khurum H; Blanco-Codesido, Montserrat; Molife, L Rhoda

    2014-06-01

    Apoptosis, a physiological process of programmed cell death, is disrupted in various malignancies. It has been exploited as an anti-cancer strategy traditionally by inducing DNA damage with chemotherapy and radiotherapy. With an increased understanding of the intrinsic and extrinsic pathways of apoptosis in recent years, novel approaches of targeting the apoptotic pathways have been tested in pre-clinical and clinical models. There are several early phase clinical trials investigating the therapeutic role of pro-apoptotic agents, both as single agents and in combination. In this review, we examine such treatment strategies, detailing the various compounds currently under clinical investigation, their potential roles in cancer therapeutics, and discussing approaches to their optimal use in the clinic.

  11. The mitochondrial death pathway: a promising therapeutic target in diseases

    PubMed Central

    Gupta, Sanjeev; Kass, George EN; Szegezdi, Eva; Joseph, Bertrand

    2009-01-01

    The mitochondrial pathway to apoptosis is a major pathway of physiological cell death in vertebrates. The mitochondrial cell death pathway commences when apoptogenic molecules present between the outer and inner mitochondrial membranes are released into the cytosol by mitochondrial outer membrane permeabilization (MOMP). BCL-2 family members are the sentinels of MOMP in the mitochondrial apoptotic pathway; the pro-apoptotic B cell lymphoma (BCL)-2 proteins, BCL-2 associated x protein and BCL-2 antagonist killer 1 induce MOMP whereas the anti-apoptotic BCL-2 proteins, BCL-2, BCL-xl and myeloid cell leukaemia 1 prevent MOMP from occurring. The release of pro-apoptotic factors such as cytochrome c from mitochondria leads to formation of a multimeric complex known as the apoptosome and initiates caspase activation cascades. These pathways are important for normal cellular homeostasis and play key roles in the pathogenesis of many diseases. In this review, we will provide a brief overview of the mitochondrial death pathway and focus on a selection of diseases whose pathogenesis involves the mitochondrial death pathway and we will examine the various pharmacological approaches that target this pathway. PMID:19220575

  12. Costunolide induces G1/S phase arrest and activates mitochondrial-mediated apoptotic pathways in SK-MES 1 human lung squamous carcinoma cells

    PubMed Central

    HUA, PEIYAN; ZHANG, GUANGXIN; ZHANG, YIFAN; SUN, MEI; CUI, RANJI; LI, XIN; LI, BINGJIN; ZHANG, XINGYI

    2016-01-01

    Despite the availability of several therapeutic options, a safer and more effective modality strategy is required for the treatment of lung cancer. Costunolide, a sesquiterpene lactone which isolated from the Saussurea lappa, has potent anticancer properties. In the present study, the effects of costunolide on cell viability, the cell cycle and apoptosis in SK-MES-1 human lung squamous carcinoma cells were investigated. Costunolide induced morphological changes and inhibited growth of SK-MES-1 cells growth. Flow cytometric analysis data demonstrated that costunolide significantly induced apoptosis of SK-MES-1 cells and induced cell cycle arrest at G1/S phase in a dose-dependent manner. Through upregulation in the expression of p53 and Bax, and downregulation in the expression of Bcl-2 and activation of caspase-3, costunolide-induced apoptosis was confirmed by western blot analysis. In addition, the significant loss of mitochondrial membrane potential indicated that costunolide may induce apoptosis via the mitochondria-dependent pathway in SK-MES-1 cells. These results highlight the potential effects of costunolide as an anti-cancer agent in a human lung squamous carcinoma cell line. PMID:27073552

  13. Costunolide induces G1/S phase arrest and activates mitochondrial-mediated apoptotic pathways in SK-MES 1 human lung squamous carcinoma cells.

    PubMed

    Hua, Peiyan; Zhang, Guangxin; Zhang, Yifan; Sun, Mei; Cui, Ranji; Li, Xin; Li, Bingjin; Zhang, Xingyi

    2016-04-01

    Despite the availability of several therapeutic options, a safer and more effective modality strategy is required for the treatment of lung cancer. Costunolide, a sesquiterpene lactone which isolated from the Saussurea lappa, has potent anticancer properties. In the present study, the effects of costunolide on cell viability, the cell cycle and apoptosis in SK-MES-1 human lung squamous carcinoma cells were investigated. Costunolide induced morphological changes and inhibited growth of SK-MES-1 cells growth. Flow cytometric analysis data demonstrated that costunolide significantly induced apoptosis of SK-MES-1 cells and induced cell cycle arrest at G1/S phase in a dose-dependent manner. Through upregulation in the expression of p53 and Bax, and downregulation in the expression of Bcl-2 and activation of caspase-3, costunolide-induced apoptosis was confirmed by western blot analysis. In addition, the significant loss of mitochondrial membrane potential indicated that costunolide may induce apoptosis via the mitochondria-dependent pathway in SK-MES-1 cells. These results highlight the potential effects of costunolide as an anti-cancer agent in a human lung squamous carcinoma cell line.

  14. Zinc ferrite nanoparticles activate IL-1b, NFKB1, CCL21 and NOS2 signaling to induce mitochondrial dependent intrinsic apoptotic pathway in WISH cells.

    PubMed

    Saquib, Quaiser; Al-Khedhairy, Abdulaziz A; Ahmad, Javed; Siddiqui, Maqsood A; Dwivedi, Sourabh; Khan, Shams T; Musarrat, Javed

    2013-12-01

    The present study has demonstrated the translocation of zinc ferrite nanoparticles (ZnFe2O4-NPs) into the cytoplasm of human amnion epithelial (WISH) cells, and the ensuing cytotoxicity and genetic damage. The results suggested that in situ NPs induced oxidative stress, alterations in cellular membrane and DNA strand breaks. The [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) and neutral red uptake (NRU) cytotoxicity assays indicated 64.48 ± 1.6% and 50.73 ± 2.1% reduction in cell viability with 100 μg/ml of ZnFe2O4-NPs exposure. The treated WISH cells exhibited 1.2-fold higher ROS level with 0.9-fold decline in membrane potential (ΔΨm) and 7.4-fold higher DNA damage after 48h of ZnFe2O4-NPs treatment. Real-time PCR (qPCR) analysis of p53, CASP 3 (caspase-3), and bax genes revealed 5.3, 1.6, and 14.9-fold upregulation, and 0.18-fold down regulation of bcl 2 gene vis-à-vis untreated control. RT(2) Profiler™ PCR array data elucidated differential up-regulation of mRNA transcripts of IL-1b, NFKB1, NOS2 and CCL21 genes in the range of 1.5 to 3.7-folds. The flow cytometry based cell cycle analysis suggested the transfer of 15.2 ± 2.1% (p<0.01) population of ZnFe2O4-NPs (100 μg/ml) treated cells into apoptotic phase through intrinsic pathway. Over all, the data revealed the potential of ZnFe2O4-NPs to induce cellular and genetic toxicity in cells of placental origin. Thus, the significant ROS production, reduction in ΔΨm, DNA damage, and activation of genes linked to inflammation, oxidative stress, proliferation, DNA damage and repair could serve as the predictive toxicity and stress markers for ecotoxicological assessment of ZnFe2O4-NPs induced cellular and genetic damage.

  15. The Modulation of Apoptotic Pathways by Gammaherpesviruses

    PubMed Central

    Banerjee, Shuvomoy; Uppal, Timsy; Strahan, Roxanne; Dabral, Prerna; Verma, Subhash C.

    2016-01-01

    Apoptosis or programmed cell death is a tightly regulated process fundamental for cellular development and elimination of damaged or infected cells during the maintenance of cellular homeostasis. It is also an important cellular defense mechanism against viral invasion. In many instances, abnormal regulation of apoptosis has been associated with a number of diseases, including cancer development. Following infection of host cells, persistent and oncogenic viruses such as the members of the Gammaherpesvirus family employ a number of different mechanisms to avoid the host cell’s “burglar” alarm and to alter the extrinsic and intrinsic apoptotic pathways by either deregulating the expressions of cellular signaling genes or by encoding the viral homologs of cellular genes. In this review, we summarize the recent findings on how gammaherpesviruses inhibit cellular apoptosis via virus-encoded proteins by mediating modification of numerous signal transduction pathways. We also list the key viral anti-apoptotic proteins that could be exploited as effective targets for novel antiviral therapies in order to stimulate apoptosis in different types of cancer cells. PMID:27199919

  16. Cytosolic pro-apoptotic SPIKE induces mitochondrial apoptosis in cancer.

    PubMed

    Nikolic, Ivana; Kastratovic, Tatjana; Zelen, Ivanka; Zivanovic, Aleksandar; Arsenijevic, Slobodan; Mitrovic, Marina

    2010-04-30

    Proteins of the BCL-2 family are important regulators of apoptosis. The BCL-2 family includes three main subgroups: the anti-apoptotic group, such as BCL-2, BCL-XL, BCL-W, and MCL-1; multi-domain pro-apoptotic BAX, BAK; and pro-apoptotic "BH3-only" BIK, PUMA, NOXA, BID, BAD, and SPIKE. SPIKE, a rare pro-apoptotic protein, is highly conserved throughout the evolution, including Caenorhabditis elegans, whose expression is downregulated in certain tumors, including kidney, lung, and breast. In the literature, SPIKE was proposed to interact with BAP31 and prevent BCL-XL from binding to BAP31. Here, we utilized the Position Weight Matrix method to identify SPIKE to be a BH3-only pro-apoptotic protein mainly localized in the cytosol of all cancer cell lines tested. Overexpression of SPIKE weakly induced apoptosis in comparison to the known BH3-only pro-apoptotic protein BIK. SPIKE promoted mitochondrial cytochrome c release, the activation of caspase 3, and the caspase cleavage of caspase's downstream substrates BAP31 and p130CAS. Although the informatics analysis of SPIKE implicates this protein as a member of the BH3-only BCL-2 subfamily, its role in apoptosis remains to be elucidated.

  17. Unravelling mitochondrial pathways to Parkinson's disease

    PubMed Central

    Celardo, I; Martins, L M; Gandhi, S

    2014-01-01

    Mitochondria are essential for cellular function due to their role in ATP production, calcium homeostasis and apoptotic signalling. Neurons are heavily reliant on mitochondrial integrity for their complex signalling, plasticity and excitability properties, and to ensure cell survival over decades. The maintenance of a pool of healthy mitochondria that can meet the bioenergetic demands of a neuron, is therefore of critical importance; this is achieved by maintaining a careful balance between mitochondrial biogenesis, mitochondrial trafficking, mitochondrial dynamics and mitophagy. The molecular mechanisms that underlie these processes are gradually being elucidated. It is widely recognized that mitochondrial dysfunction occurs in many neurodegenerative diseases, including Parkinson's disease. Mitochondrial dysfunction in the form of reduced bioenergetic capacity, increased oxidative stress and reduced resistance to stress, is observed in several Parkinson's disease models. However, identification of the recessive genes implicated in Parkinson's disease has revealed a common pathway involving mitochondrial dynamics, transport, turnover and mitophagy. This body of work has led to the hypothesis that the homeostatic mechanisms that ensure a healthy mitochondrial pool are key to neuronal function and integrity. In this paradigm, impaired mitochondrial dynamics and clearance result in the accumulation of damaged and dysfunctional mitochondria, which may directly induce neuronal dysfunction and death. In this review, we consider the mechanisms by which mitochondrial dysfunction may lead to neurodegeneration. In particular, we focus on the mechanisms that underlie mitochondrial homeostasis, and discuss their importance in neuronal integrity and neurodegeneration in Parkinson's disease. LINKED ARTICLES This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph

  18. Evaluating the Protective Effects and Mechanisms of Diallyl Disulfide on Interlukin-1β-Induced Oxidative Stress and Mitochondrial Apoptotic Signaling Pathways in Cultured Chondrocytes.

    PubMed

    Hosseinzadeh, Azam; Jafari, Davood; Kamarul, Tunku; Bagheri, Abolfazll; Sharifi, Ali M

    2017-02-07

    The protective effects and mechanisms of DADS on IL-1β-mediated oxidative stress and mitochondrial apoptosis were investigated in C28I2 human chondrocytes. The effect of various concentrations of DADS (1, 5 10, 25, 50, and 100 μM) on C28I2 cell viability was evaluated in different times (2, 4, 8, 16, and 24 h) to obtain the non-cytotoxic concentrations of drug by MTT-assay. The protective effect of non-toxic concentrations of DADS on experimentally induced oxidative stress and apoptosis by IL-1β in C28I2 was evaluated. The effects of DADS on IL-1β-induced intracellular ROS production and lipid peroxidation were detected and the proteins expression of Nrf2, Bax, Bcl-2, caspase-3, total and phosphorylated JNK, and P38 MAPKs were analyzed by Western blotting. The mRNA expression of detoxifying phase II/antioxidant enzymes including heme oxygenase-1, NAD(P)H quinine oxidoreductase, glutathione S-transferase-P1, catalase, superoxide dismutase-1, glutathione peroxidase-1, -3, -4 were evaluated by reverse transcription-polymerase chain reaction. DADS in 1, 5, 10, and 25 μM concentrations had no cytotoxic effect after 24 h. Pretreatment with DADS remarkably increased Nrf2 nuclear translocation as well as the genes expression of detoxifying phase II/antioxidant enzymes and reduced IL-1β-induced elevation of ROS, lipid peroxidation, Bax/Bcl-2 ratio, caspase-3 activation, and JNK and P38 phosphorylation. DADS could considerably reduce IL-1β-induced oxidative stress and consequent mitochondrial apoptosis, as the major mechanisms of chondrocyte cell death in an experimental model of osteoarthritis. It may be considered as natural product in protecting OA-induced cartilage damage in clinical setting. J. Cell. Biochem. 9999: 1-10, 2017. © 2017 Wiley Periodicals, Inc.

  19. Interaction of pro-apoptotic protein HGTD-P with heat shock protein 90 is required for induction of mitochondrial apoptotic cascades.

    PubMed

    Kim, Jee-Youn; Kim, Su-Mi; Ko, Jeong-Hun; Yim, Ji-Hye; Park, Jin-Hae; Park, Jae-Hoon

    2006-05-29

    HGTD-P is a hypoxia-responsive pro-apoptotic protein that transmits hypoxic signals directly to mitochondria. When overexpressed, HGTD-P induces cell death via typical mitochondrial apoptotic cascades. However, much is unknown about post-transcriptional modification and signaling networks of HGTD-P in association with cell death-regulating proteins. We performed yeast two-hybrid screening to identify the molecules involved in HGTD-P-mediated cell death pathways. In this study, we show that heat shock protein 90 physically interacts with HGTD-P and that suppression of Hsp90 activity by low concentrations of geldanamycin reduced HGTD-P-induced mitochondrial catastrophe through inhibition of mitochondrial translocation of HGTD-P.

  20. Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection.

    PubMed

    Claus, Claudia; Manssen, Lena; Hübner, Denise; Roßmark, Sarah; Bothe, Viktoria; Petzold, Alice; Große, Claudia; Reins, Mareen; Mankertz, Annette; Frey, Teryl K; Liebert, Uwe G

    2015-11-26

    Mitochondria- as well as p53-based signaling pathways are central for the execution of the intrinsic apoptotic cascade. Their contribution to rubella virus (RV)-induced apoptosis was addressed through time-specific evaluation of characteristic parameters such as permeabilization of the mitochondrial membrane and subsequent release of the pro-apoptotic proteins apoptosis-inducing factor (AIF) and cytochrome c from mitochondria. Additionally, expression and localization pattern of p53 and selected members of the multifunctional and stress-inducible cyclophilin family were examined. The application of pifithrin μ as an inhibitor of p53 shuttling to mitochondria reduced RV-induced cell death to an extent similar to that of the broad spectrum caspase inhibitor z-VAD-fmk (benzyloxycarbonyl-V-A-D-(OMe)-fmk). However, RV progeny generation was not altered. This indicates that, despite an increased survival rate of its cellular host, induction of apoptosis neither supports nor restricts RV replication. Moreover, some of the examined apoptotic markers were affected in a strain-specific manner and differed between the cell culture-adapted strains: Therien and the HPV77 vaccine on the one hand, and a clinical isolate on the other. In summary, the results presented indicate that the transcription-independent mitochondrial p53 program contributes to RV-induced apoptosis.

  1. Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection

    PubMed Central

    Claus, Claudia; Manssen, Lena; Hübner, Denise; Roßmark, Sarah; Bothe, Viktoria; Petzold, Alice; Große, Claudia; Reins, Mareen; Mankertz, Annette; Frey, Teryl K.; Liebert, Uwe G.

    2015-01-01

    Mitochondria- as well as p53-based signaling pathways are central for the execution of the intrinsic apoptotic cascade. Their contribution to rubella virus (RV)-induced apoptosis was addressed through time-specific evaluation of characteristic parameters such as permeabilization of the mitochondrial membrane and subsequent release of the pro-apoptotic proteins apoptosis-inducing factor (AIF) and cytochrome c from mitochondria. Additionally, expression and localization pattern of p53 and selected members of the multifunctional and stress-inducible cyclophilin family were examined. The application of pifithrin μ as an inhibitor of p53 shuttling to mitochondria reduced RV-induced cell death to an extent similar to that of the broad spectrum caspase inhibitor z-VAD-fmk (benzyloxycarbonyl-V-A-D-(OMe)-fmk). However, RV progeny generation was not altered. This indicates that, despite an increased survival rate of its cellular host, induction of apoptosis neither supports nor restricts RV replication. Moreover, some of the examined apoptotic markers were affected in a strain-specific manner and differed between the cell culture-adapted strains: Therien and the HPV77 vaccine on the one hand, and a clinical isolate on the other. In summary, the results presented indicate that the transcription-independent mitochondrial p53 program contributes to RV-induced apoptosis. PMID:26703711

  2. Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells

    PubMed Central

    2010-01-01

    Background Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO2) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways. Results Detailed time course studies revealed that both CB (13 nm) and TiO2(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO2 but hydrogen peroxide (H2O2) production was only involved in apoptosis induction by CB NPs. Conclusions Both CB and TiO2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs. PMID:20398356

  3. Impact of Antioxidants on Cardiolipin Oxidation in Liposomes: Why Mitochondrial Cardiolipin Serves as an Apoptotic Signal?

    PubMed Central

    Lokhmatikov, Alexey V.; Voskoboynikova, Natalia; Cherepanov, Dmitry A.; Skulachev, Maxim V.; Steinhoff, Heinz-Jürgen; Skulachev, Vladimir P.; Mulkidjanian, Armen Y.

    2016-01-01

    Molecules of mitochondrial cardiolipin (CL) get selectively oxidized upon oxidative stress, which triggers the intrinsic apoptotic pathway. In a chemical model most closely resembling the mitochondrial membrane—liposomes of pure bovine heart CL—we compared ubiquinol-10, ubiquinol-6, and alpha-tocopherol, the most widespread naturally occurring antioxidants, with man-made, quinol-based amphiphilic antioxidants. Lipid peroxidation was induced by addition of an azo initiator in the absence and presence of diverse antioxidants, respectively. The kinetics of CL oxidation was monitored via formation of conjugated dienes at 234 nm. We found that natural ubiquinols and ubiquinol-based amphiphilic antioxidants were equally efficient in protecting CL liposomes from peroxidation; the chromanol-based antioxidants, including alpha-tocopherol, were 2-3 times less efficient. Amphiphilic antioxidants, but not natural ubiquinols and alpha-tocopherol, were able, additionally, to protect the CL bilayer from oxidation by acting from the water phase. We suggest that the previously reported therapeutic efficiency of mitochondrially targeted amphiphilic antioxidants is owing to their ability to protect those CL molecules that are inaccessible to natural hydrophobic antioxidants, being trapped within respiratory supercomplexes. The high susceptibility of such occluded CL molecules to oxidation may have prompted their recruitment as apoptotic signaling molecules by nature. PMID:27313834

  4. Impact of Antioxidants on Cardiolipin Oxidation in Liposomes: Why Mitochondrial Cardiolipin Serves as an Apoptotic Signal?

    PubMed

    Lokhmatikov, Alexey V; Voskoboynikova, Natalia; Cherepanov, Dmitry A; Skulachev, Maxim V; Steinhoff, Heinz-Jürgen; Skulachev, Vladimir P; Mulkidjanian, Armen Y

    2016-01-01

    Molecules of mitochondrial cardiolipin (CL) get selectively oxidized upon oxidative stress, which triggers the intrinsic apoptotic pathway. In a chemical model most closely resembling the mitochondrial membrane-liposomes of pure bovine heart CL-we compared ubiquinol-10, ubiquinol-6, and alpha-tocopherol, the most widespread naturally occurring antioxidants, with man-made, quinol-based amphiphilic antioxidants. Lipid peroxidation was induced by addition of an azo initiator in the absence and presence of diverse antioxidants, respectively. The kinetics of CL oxidation was monitored via formation of conjugated dienes at 234 nm. We found that natural ubiquinols and ubiquinol-based amphiphilic antioxidants were equally efficient in protecting CL liposomes from peroxidation; the chromanol-based antioxidants, including alpha-tocopherol, were 2-3 times less efficient. Amphiphilic antioxidants, but not natural ubiquinols and alpha-tocopherol, were able, additionally, to protect the CL bilayer from oxidation by acting from the water phase. We suggest that the previously reported therapeutic efficiency of mitochondrially targeted amphiphilic antioxidants is owing to their ability to protect those CL molecules that are inaccessible to natural hydrophobic antioxidants, being trapped within respiratory supercomplexes. The high susceptibility of such occluded CL molecules to oxidation may have prompted their recruitment as apoptotic signaling molecules by nature.

  5. The apoptotic pathways effect of fine particulate from cooking oil fumes in primary fetal alveolar type II epithelial cells.

    PubMed

    Che, Zhen; Liu, Ying; Chen, Yanyan; Cao, Jiyu; Liang, Chunmei; Wang, Lei; Ding, Rui

    2014-02-01

    Apoptosis occurs along three major pathways: (i) an extrinsic pathway, mediated by death receptors; (ii) an intrinsic pathway centered on mitochondria; and (iii) an ER-stress pathway. We investigated the apoptotic pathway effects of cooking oil fumes (COF) in fetal lung type II-like epithelium cells (AEC II). Exposure to COF caused up-regulation of the pro-apoptotic protein Bax and down-regulation of the anti-apoptotic protein Bcl-2. COF induced the mitochondrial permeability transition, an early event in apoptosis; cytochrome c was translocated from the mitochondria to the cytoplasm and nucleus. Caspase-9 and caspase-3 were activated, as a consequence of the mitochondrial permeability transition. The death receptor apoptotic pathway was triggered by COF, as indicated by a change in Fas expression, resulting in increased caspase-8 content. COF exposure arrested the cell cycle the at G0-G1 phase. In summary, COF can lead to apoptosis via mitochondrial and death receptor pathways in AEC II cells.

  6. The inflammatory role of phagocyte apoptotic pathways in rheumatic diseases

    PubMed Central

    Cuda, Carla M.; Pope, Richard M.; Perlman, Harris

    2017-01-01

    Rheumatoid arthritis affects nearly 1% of the world's population and is a debilitating autoimmune condition that can result in joint destruction. During the past decade, inflammatory functions have been described for signalling molecules classically involved in apoptotic and non-apoptotic death pathways, including but not limited to toll-like receptor signalling, inflammasome activation, cytokine production, macrophage polarization and antigen citrullination. In light of these remarkable advances in the understanding of inflammatory mechanisms of the death machinery, this review provides a snapshot of the available evidence implicating death pathways, especially within the phagocyte populations of the innate immune system, in the perpetuation of rheumatoid arthritis. Elevated levels of signalling mediators of both the extrinsic and intrinsic apoptotic as well as the autophagy death pathways are observed in the joints of patients with rheumatoid arthritis. Furthermore, in rheumatoid arthritis patients, risk polymorphisms are present in signalling molecules of the extrinsic apoptotic and autophagy death pathways. Although research into the mechanisms underlying these death pathways has made considerable progress, this review highlights areas where further investigation is particularly needed. This exploration is critical, as new discoveries in this field could lead to the development of novel therapeutic targets for rheumatoid arthritis and other rheumatic diseases. PMID:27549026

  7. Regulation of Intrinsic and Extrinsic Apoptotic Pathways in Osteosarcoma Cells Following Oleandrin Treatment

    PubMed Central

    Ma, Yunlong; Zhu, Bin; Yong, Lei; Song, Chunyu; Liu, Xiao; Yu, Huilei; Wang, Peng; Liu, Zhongjun; Liu, Xiaoguang

    2016-01-01

    Our previous study has reported the anti-tumor effect of oleandrin on osteosarcoma (OS) cells. In the current study, we mainly explored its potential regulation on intrinsic and extrinsic apoptotic pathway in OS cells. Cells apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected using fluorescence staining and flow cytometry. Caspase-3 activity was detected using a commercial kit. The levels of cytoplasmic cytochrome c, mitochondrial cytochrome c, bcl-2, bax, caspase-9, Fas, FasL, caspase-8 and caspase-3 were detected by Western blotting. z-VAD-fmk was applied to block both intrinsic and extrinsic apoptosis pathways, and cells apoptosis was also tested. Furthermore, we used z-LEHD-fmk and Fas blocking antibody to inhibit intrinsic and extrinsic pathways, separately, and the selectivity of oleandrin on these pathways was explored. Results showed that oleandrin induced the apoptosis of OS cells, which was accompanied by an increase in ROS and a decrease in MMP. Furthermore, cytochrome c level was reduced in mitochondria but elevated in the cytoplasm. Caspase-3 activity was enhanced by oleandrin in a concentration- and time-dependent manner. Oleandrin also down-regulated the expression of bcl-2, but up-regulated bax, caspase-9, Fas, FasL, caspase-8 and caspase-3. In addition, the suppression of both apoptotic pathways by z-VAD-fmk greatly reverted the oleandrin-induced apoptosis. Moreover, the suppression of one pathway by a corresponding inhibitor did not affect the regulation of oleandrin on another pathway. Taken together, we concluded that oleandrin induced apoptosis of OS cells via activating both intrinsic and extrinsic apoptotic pathways. PMID:27886059

  8. The effects of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function after glutamate excitotoxicity.

    PubMed

    Wang, Xiaowan; Li, Hailong; Ding, Shinghua

    2014-11-07

    NAD+ is an essential co-enzyme for cellular energy metabolism and is also involved as a substrate for many cellular enzymatic reactions. It has been shown that NAD+ has a beneficial effect on neuronal survival and brain injury in in vitro and in vivo ischemic models. However, the effect of NAD+ on mitochondrial biogenesis and function in ischemia has not been well investigated. In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function. Our results show that supplementation of NAD+ could effectively reduce apoptotic neuronal death, and apoptotic inducing factor translocation after neurons were challenged with excitotoxic glutamate stimulation. Using different approaches including confocal imaging, mitochondrial DNA measurement and Western blot analysis of PGC-1 and NRF-1, we also found that NAD+ could significantly attenuate glutamate-induced mitochondrial fragmentation and the impairment of mitochondrial biogenesis. Furthermore, NAD+ treatment effectively inhibited mitochondrial membrane potential depolarization and NADH redistribution after excitotoxic glutamate stimulation. Taken together, our results demonstrated that NAD+ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity. Our findings provide insights into potential neuroprotective strategies in ischemic stroke.

  9. Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia

    PubMed Central

    Wang, Xiaowan; Li, Hailong; Ding, Shinghua

    2016-01-01

    We previously demonstrated that Pre-B-cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis pathway, plays a brain and neuronal protective role in ischemic stroke. In this study, we further investigated the mechanism of its neuroprotective effect after ischemia in the primary cultured mouse cortical neurons. Using apoptotic cell death assay, fluorescent imaging, molecular biology, mitochondrial biogenesis measurements and Western blotting analysis, our results show that the overexpression of PBEF in neurons can significantly promote neuronal survival, reduce the translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei and inhibit the activation of capase-3 after glutamate-induced excitotoxicity. We further found that the overexpression of PBEF can suppress glutamate-induced mitochondrial fragmentation, the loss of mitochondrial DNA (mtDNA) content and the reduction of PGC-1 and NRF-1 expressions. Furthermore, these beneficial effects by PBEF are dependent on its enzymatic activity of NAD+ synthesis. In summary, our study demonstrated that PBEF ameliorates ischemia-induced neuronal death through inhibiting caspase-dependent and independent apoptotic signaling pathways and suppressing mitochondrial damage and dysfunction. Our study provides novel insights into the mechanisms underlying the neuroprotective effect of PBEF, and helps to identify potential targets for ischemic stroke therapy. PMID:27576732

  10. Induction of discrete apoptotic pathways by bromo-substituted indirubin derivatives in invasive breast cancer cells

    SciTech Connect

    Nicolaou, Katerina A.; Liapis, Vasilis; Evdokiou, Andreas; Constantinou, Constantina; Magiatis, Prokopios; Skaltsounis, Alex L.; Koumas, Laura; Costeas, Paul A.; Constantinou, Andreas I.

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer The effects of 6BIO and 7BIO are evaluated against five breast cancer cell lines. Black-Right-Pointing-Pointer 6BIO induces a caspase dependent apoptotic effect via the intrinsic pathway. Black-Right-Pointing-Pointer 7BIO promotes G{sub 2}/M cells cycle arrest. Black-Right-Pointing-Pointer 7BIO triggers a caspase-8 mediated apoptotic pathway. Black-Right-Pointing-Pointer 7BIO triggers and a caspase independent pathway. -- Abstract: Indirubin derivatives gained interest in recent years for their anticancer and antimetastatic properties. The objective of the present study was to evaluate and compare the anticancer properties of the two novel bromo-substituted derivatives 6-bromoindirubin-3 Prime -oxime (6BIO) and 7-bromoindirubin-3 Prime -oxime (7BIO) in five different breast cancer cell lines. Cell viability assays identified that 6BIO and 7BIO are most effective in preventing the proliferation of the MDA-MB-231-TXSA breast cancer cell line from a total of five breast cancer cell lined examined. In addition it was found that the two compounds induce apoptosis via different mechanisms. 6BIO induces caspase-dependent programmed cell death through the intrinsic (mitochondrial) caspase-9 pathway. 7BIO up-regulates p21 and promotes G{sub 2}/M cell cycle arrest which is subsequently followed by the activation of two different apoptotic pathways: (a) a pathway that involves the upregulation of DR4/DR5 and activation of caspase-8 and (b) a caspase independent pathway. In conclusion, this study provides important insights regarding the molecular pathways leading to cell cycle arrest and apoptosis by two indirubin derivatives that can find clinical applications in targeted cancer therapeutics.

  11. The mitochondrial pathway of anesthetic isoflurane-induced apoptosis.

    PubMed

    Zhang, Yiying; Dong, Yuanlin; Wu, Xu; Lu, Yan; Xu, Zhipeng; Knapp, Andrew; Yue, Yun; Xu, Tiejun; Xie, Zhongcong

    2010-02-05

    The common inhalation anesthetic isoflurane has been shown to induce apoptosis, which then leads to accumulation of beta-amyloid protein, the hallmark feature of Alzheimer disease neuropathogenesis. The underlying molecular mechanism of the isoflurane-induced apoptosis is largely unknown. We, therefore, set out to assess whether isoflurane can induce apoptosis by regulating Bcl-2 family proteins, enhancing reactive oxygen species (ROS) accumulation, and activating the mitochondrial pathway of apoptosis. We performed these studies in cultured cells, primary neurons, and mice. Here we show for the first time that treatment with 2% isoflurane for 6 h can increase pro-apoptotic factor Bax levels, decrease anti-apoptotic factor Bcl-2 levels, increase ROS accumulation, facilitate cytochrome c release from the mitochondria to the cytosol, induce activation of caspase-9 and caspase-3, and finally cause apoptosis as compared with the control condition. We have further found that isoflurane can increase the mRNA levels of Bax and reduce the mRNA levels of Bcl-2. The isoflurane-induced ROS accumulation can be attenuated by the intracellular calcium chelator BAPTA. Finally, the anesthetic desflurane does not induce activation of mitochondrial pathway of apoptosis. These results suggest that isoflurane may induce apoptosis through Bcl-2 family proteins- and ROS-associated mitochondrial pathway of apoptosis. These findings, which have identified at least partially the molecular mechanism by which isoflurane induces apoptosis, will promote more studies aimed at studying the potential neurotoxic effects of anesthetics.

  12. FOXO3a governs early and late apoptotic endothelial programs during elevated glucose through mitochondrial and caspase signaling.

    PubMed

    Hou, Jinling; Chong, Zhao Zhong; Shang, Yan Chen; Maiese, Kenneth

    2010-06-10

    Mechanisms that preserve endothelial cell (EC) integrity remain elusive, but are critical for new strategies directed against endocrine disorders such as diabetes mellitus (DM). Here we demonstrate in primary cerebral ECs with a clinically relevant model of elevated d-glucose that Akt1 and the post-translational modification and subcellular trafficking of the forkhead transcription factor FoxO3a are critical for early apoptotic membrane signaling and subsequent degradation of nuclear DNA. FoxO3a also directly governs apoptotic mitochondrial signal transduction pathways, since gene knockdown of FoxO3a prevents mitochondrial membrane depolarization as well as the release of cytochrome c. Control of this apoptotic cascade extends to the rapid and progressive activation of caspases. The presence of FoxO3a is necessary for cleaved (active) caspase 1 and 3 expression, since loss of FoxO3a abrogates the induction of caspase activity. Our work identifies Akt1, FoxO3a and closely aligned pathways as key therapeutic targets during impaired glucose tolerance and DM.

  13. Early apoptotic vascular signaling is determined by Sirt1 through nuclear shuttling, forkhead trafficking, bad, and mitochondrial caspase activation.

    PubMed

    Hou, Jinling; Chong, Zhao Zhong; Shang, Yan Chen; Maiese, Kenneth

    2010-05-01

    Complications of diabetes mellitus (DM) weigh heavily upon the endothelium that ultimately affect multiple organ systems. These concerns call for innovative treatment strategies that employ molecular pathways responsible for cell survival and longevity. Here we show in a clinically relevant model of DM with elevated D-glucose that endothelial cell (EC) SIRT1 is vital for the prevention of early membrane apoptotic phosphatidylserine externalization and subsequent DNA degradation supported by studies with modulation of SIRT1 activity and gene knockdown of SIRT1. Furthermore, during elevated D-glucose exposure, we show that SIRT1 is sequestered in the cytoplasm of ECs, but specific activation of SIRT1 shuttles the protein to the nucleus to allow for cytoprotection. The ability of SIRT1 to avert apoptosis employs the activation of protein kinase B (Akt1), the post-translational phosphorylation of the forkhead member FoxO3a, the blocked trafficking of FoxO3a to the nucleus, and the inhibition of FoxO3a to initiate a "pro-apoptotic" program as shown by complimentary gene knockdown studies of FoxO3a. Vascular apoptotic oversight by SIRT1 extends to the direct modulation of mitochondrial membrane permeability, cytochrome c release, Bad activation, and caspase 1 and 3 activation, since inhibition of SIRT1 activity and gene knockdown of SIRT1 significantly accentuate cascade progression while SIRT1 activation abrogates these apoptotic elements. Our work identifies vascular SIRT1 and its control over early apoptotic membrane signaling, Akt1 activation, post-translational modification and trafficking of FoxO3a, mitochondrial permeability, Bad activation, and rapid caspase induction as new avenues for the treatment of vascular complications during DM.

  14. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand.

    PubMed

    Saralamma, Venu Venkatarame Gowda; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

    2015-09-18

    Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies' results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer.

  15. Colon cancer cell chemosensitisation by fish oil emulsion involves apoptotic mitochondria pathway.

    PubMed

    Granci, Virginie; Cai, Fang; Lecumberri, Elena; Clerc, Aurélie; Dupertuis, Yves M; Pichard, Claude

    2013-04-14

    Adjuvant use of safe compounds with anti-tumour properties has been proposed to improve cancer chemotherapy outcome. We aimed to investigate the effects of fish oil emulsion (FOE) rich in n-3 PUFA with the standard chemotherapeutic agents 5-fluorouracil (5-FU), oxaliplatin (OX) or irinotecan (IRI) on two human colorectal adenocarcinoma cells with different genetic backgrounds. The HT-29 (Bax+/+) and LS174T (Bax-/-) cells were co-treated for 24-72 h with 1 μm-5-FU, 1 μm-OX or 10 μm-IRI and/or FOE dilution corresponding to 24 μm-EPA and 20·5 μm-DHA. Soyabean oil emulsion (SOE) was used as isoenergetic and isolipid control. Cell viability, apoptosis and nuclear morphological changes were evaluated by cytotoxic colorimetric assay, flow cytometry analysis with annexin V and 4',6'-diamidino-2-phenylindole staining, respectively. A cationic fluorescent probe was used to evaluate mitochondrial dysfunction, and protein expression involved in mitochondrial apoptosis was determined by Western blot. In contrast to SOE, co-treatment with FOE enhanced significantly the pro-apoptotic and cytotoxic effects of 5-FU, OX or IRI in HT-29 but not in LS174T cells (two-way ANOVA, P <0.01). These results were confirmed by the formation of apoptotic bodies in HT-29 cells. A significant increase in mitochondrial membrane depolarisation was observed after the combination of 5-FU or IRI with FOE in HT-29 but not in LS174T cells (P <0.05). Co-administration of FOE with the standard agents, 5-FU, OX and IRI, could be a good alternative to increase the efficacy of chemotherapeutic protocols through a Bax-dependent mitochondrial pathway.

  16. Effect of chronic contractile activity on SS and IMF mitochondrial apoptotic susceptibility in skeletal muscle.

    PubMed

    Adhihetty, Peter J; Ljubicic, Vladimir; Hood, David A

    2007-03-01

    Chronic contractile activity of skeletal muscle induces an increase in mitochondria located in proximity to the sarcolemma [subsarcolemmal (SS)] and in mitochondria interspersed between the myofibrils [intermyofibrillar (IMF)]. These are energetically favorable metabolic adaptations, but because mitochondria are also involved in apoptosis, we investigated the effect of chronic contractile activity on mitochondrially mediated apoptotic signaling in muscle. We hypothesized that chronic contractile activity would provide protection against mitochondrially mediated apoptosis despite an elevation in the expression of proapoptotic proteins. To induce mitochondrial biogenesis, we chronically stimulated (10 Hz; 3 h/day) rat muscle for 7 days. Chronic contractile activity did not alter the Bax/Bcl-2 ratio, an index of apoptotic susceptibility, and did not affect manganese superoxide dismutase levels. However, contractile activity increased antiapoptotic 70-kDa heat shock protein and apoptosis repressor with a caspase recruitment domain by 1.3- and 1.4-fold (P<0.05), respectively. Contractile activity elevated SS mitochondrial reactive oxygen species (ROS) production 1.4- and 1.9-fold (P<0.05) during states IV and III respiration, respectively, whereas IMF mitochondrial state IV ROS production was suppressed by 28% (P<0.05) and was unaffected during state III respiration. Following stimulation, exogenous ROS treatment produced less cytochrome c release (25-40%) from SS and IMF mitochondria, and also reduced apoptosis-inducing factor release (approximately 30%) from IMF mitochondria, despite higher inherent cytochrome c and apoptosis-inducing factor expression. Chronic contractile activity did not alter mitochondrial permeability transition pore (mtPTP) components in either subfraction. However, SS mitochondria exhibited a significant increase in the time to Vmax of mtPTP opening. Thus, chronic contractile activity induces predominantly antiapoptotic adaptations in both

  17. Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

    PubMed Central

    Nantajit, Danupon; Fan, Ming; Duru, Nadire; Wen, Yunfei; Reed, John C.; Li, Jian Jian

    2010-01-01

    The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53. PMID:20808790

  18. HIV-1 promonocytic and lymphoid cell lines: an in vitro model of in vivo mitochondrial and apoptotic lesion.

    PubMed

    Morén, Constanza; González-Casacuberta, Ingrid; Álvarez-Fernández, Carmen; Bañó, Maria; Catalán-Garcia, Marc; Guitart-Mampel, Mariona; Juárez-Flores, Diana Luz; Tobías, Ester; Milisenda, José; Cardellach, Francesc; Gatell, Josep Maria; Sánchez-Palomino, Sonsoles; Garrabou, Glòria

    2017-02-01

    To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV-1)-infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro-mitochondrial or anti-apoptotic strategies as in vitro cell platforms to deal with HIV-infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt-PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial-nuclear encoded subunits II-IV of cytochrome-c-oxidase (COXII-COXIV), respectively, as well as mitochondrial apoptotic events [voltage-dependent-anion-channel-1(VDAC-1)-content and caspase-9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1-assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC-1 and caspase-9 content were sharply decreased in both chronic HIV-1-infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV-chronicity, to test novel therapeutic options or

  19. Ethanol-induced oxidative stress precedes mitochondrially mediated apoptotic death of cultured fetal cortical neurons.

    PubMed

    Ramachandran, Vinitha; Watts, Lora Talley; Maffi, Shivani Kaushal; Chen, Juanjuan; Schenker, Steven; Henderson, George

    2003-11-15

    In utero ethanol exposure elicits apoptotic cell death in the fetal brain, and this may be mediated by oxidative stress. Our studies utilize cultured fetal rat cortical neurons and illustrate that ethanol elicits a rapid onset of oxidative stress, which culminates in mitochondrially mediated apoptotic cell death. Cells exposed to ethanol (2.5 mg/ml) remained attached to their polylysine matrix during a 24-hr exposure, but they exhibited distinct signs of oxidative stress, decreased viability, and apoptosis. Confocal microscopy of live cortical neurons pretreated with dichlorodihydrofluorescein diacetate demonstrated an increase in reactive oxygen species (ROS) within 5 min of ethanol exposure. The levels of ROS further increased by 58% within 1 hr (P <.05) and by 82% within 2 hr (P <.05), accompanied by increases of mitochondrial 4-hydroxynonenal (HNE). These early events were followed by decreased trypan blue exclusion of 10% to 32% (P <.05) at the 6- to 24-hr time points, respectively. This culminates in apoptotic death, with increases of Annexin V binding of 43%, 89%, 123%, and 238%, at 2, 6, 12, and 24 hr of ethanol treatment, respectively, as well as DNA fragmentation increases of 50% and 65% by 12 and 24 hr, respectively. Release of cytochrome c by mitochondria increased by 53% at 6 hr of exposure (P <.05), concomitant with activation of caspase 3 (52% at 12 hr, P <.05). Pretreatment with N-acetylcysteine increased cellular glutathione and prevented apoptosis. These studies provide a time line illustrating that oxidative stress and formation of a proapoptotic lipid peroxidation product, HNE, precede a cascade of mitochondrially mediated events in cultured fetal cortical neurons, culminating in apoptotic death. The prevention of apoptosis by augmentation of glutathione stores also strongly supports a role for oxidative stress in ethanol-mediated apoptotic death of fetal cortical neurons.

  20. Mitochondrial staining allows robust elimination of apoptotic and damaged cells during cell sorting.

    PubMed

    Barteneva, Natasha S; Ponomarev, Eugeny D; Tsytsykova, Alla; Armant, Myriam; Vorobjev, Ivan A

    2014-04-01

    High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE(+) cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations.

  1. Expression of FADD and cFLIPL balances mitochondrial integrity and redox signaling to substantiate apoptotic cell death.

    PubMed

    Ranjan, Kishu; Pathak, Chandramani

    2016-11-01

    FADD and cFLIP both are pivotal components of death receptor signaling. The cellular signaling of apoptosis accomplished with death receptors and mitochondria follows independent pathways for cell death. FADD and cFLIP both have an important role in the regulation of apoptotic and non-apoptotic functions. Dysregulated expression of FADD and cFLIP is associated with resistance to apoptosis in cancer cells. Mitochondria are known to play critical role in maintaining cellular respiration and homeostasis in the cells as well as transduces various signals to determine the fate of cell death. However, involvement of FADD and cFLIP in regulation of mitochondrial integrity and programmed cell death signaling to define the fate of cells remains elusive. In the present study, we explored that, induced expression of FADD challenges the mitochondrial integrity and pulverizes the membrane potential by altering the expression of Bcl-2 and cytochrome c. In contrast, mutant of FADD was unable to affect the mitochondrial integrity. Interestingly, expression of FADD and cFLIP helps to balance redox potential by regulating the anti-oxidant levels. Further, we noticed that, knockdown of cFLIPL and induced expression of FADD rapidly accumulate intracellular ROS accompanied by JNK1 activation to substantiate apoptosis. Notably, the ectopic expression of cFLIPL resists the sensitivity of cancer cells against apoptosis inducers Etoposide and HA14-1. Altogether, our findings suggest that FADD and cFLIPL are important modulators of mitochondrial-associated apoptosis apart from the death receptor signaling.

  2. Effects of glycerol on apoptotic signaling pathways during boar spermatozoa cryopreservation.

    PubMed

    Zeng, Changjun; Tang, Keyi; He, Lian; Peng, Wenpei; Ding, Li; Fang, Donghui; Zhang, Yan

    2014-06-01

    Artificial insemination (AI) with post-thawed boar spermatozoa results in low farrowing rates and reduced litter sizes mainly due to cryoinjury or damages to spermatozoa during cryopreservation. Low viability and motility of post-thawed boar spermatozoa are highly associated with apoptosis during cryopreservation. Although glycerol is widely used a cryoprotectant (CPA) for boar spermatozoa cryopreservation, the mechanism and relationship between glycerol and apoptosis-related gene expression needs to be clarified. In this study, we treated boar spermatozoa with different concentrations of glycerol in lactose egg yolk (LEY) extender to evaluate the apoptosis-related gene expression and protease activities of caspases. These results show that: (1) low concentrations of glycerol (2% and 3%) were more suitable for boar spermatozoa cryopreservation; (2) apoptosis-related genes involved in intrinsic mitochondrial and extrinsic death receptor apoptotic signaling pathways were widely expressed in different concentrations of glycerol treated boar spermatozoa; (3) there was a significant positive correlation (r=0.840, P=0.037) between the percentage of Annexin V(+)/PI(+) staining spermatozoa and caspase-6/9 protease activity. In conclusion, 2% and 3% glycerol have the best anti-apoptotic effects, and the expression of Fas/FasL and Bcl-2/Bax have a strong correlation with spermatozoa parameters.

  3. Activation of intrinsic apoptotic signaling pathway in cancer cells by Cymbopogon citratus polysaccharide fractions.

    PubMed

    Thangam, Ramar; Sathuvan, Malairaj; Poongodi, Arasu; Suresh, Veeraperumal; Pazhanichamy, Kalailingam; Sivasubramanian, Srinivasan; Kanipandian, Nagarajan; Ganesan, Nalini; Rengasamy, Ramasamy; Thirumurugan, Ramasamy; Kannan, Soundarapandian

    2014-07-17

    Essential oils of Cymbopogon citratus were already reported to have wide ranging medical and industrial applications. However, information on polysaccharides from the plant and their anticancer activities are limited. In the present study, polysaccharides from C. citratus were extracted and fractionated by anion exchange and gel filtration chromatography. Two different polysaccharide fractions such as F1 and F2 were obtained, and these fractions were found to have distinct acidic polysaccharides as characterized by their molecular weight and sugar content. NMR spectral analysis revealed the presence of (1→4) linked b-d-Xylofuranose moiety in these polysaccharides. Using these polysaccharide fractions F1 and F2, anti-inflammatory and anticancer activities were evaluated against cancer cells in vitro and the mechanism of action of the polysaccharides in inducing apoptosis in cancer cells via intrinsic pathway was also proposed. Two different reproductive cancer cells such as Siha and LNCap were employed for in vitro studies on cytotoxicity, induction of apoptosis and apoptotic DNA fragmentation, changes in mitochondrial membrane potential, and profiles of gene and protein expression in response to treatment of cells by the polysaccharide fractions. These polysaccharide fractions exhibited potential cytotoxic and apoptotic effects on carcinoma cells, and they induced apoptosis in these cells through the events of up-regulation of caspase 3, down-regulation of bcl-2 family genes followed by cytochrome c release.

  4. Ebola Virus Does Not Block Apoptotic Signaling Pathways

    PubMed Central

    Olejnik, Judith; Alonso, Jesus; Schmidt, Kristina M.; Yan, Zhen; Wang, Wei; Marzi, Andrea; Ebihara, Hideki; Yang, Jinghua; Patterson, Jean L.; Ryabchikova, Elena

    2013-01-01

    Since viruses rely on functional cellular machinery for efficient propagation, apoptosis is an important mechanism to fight viral infections. In this study, we sought to determine the mechanism of cell death caused by Ebola virus (EBOV) infection by assaying for multiple stages of apoptosis and hallmarks of necrosis. Our data indicate that EBOV does not induce apoptosis in infected cells but rather leads to a nonapoptotic form of cell death. Ultrastructural analysis confirmed necrotic cell death of EBOV-infected cells. To investigate if EBOV blocks the induction of apoptosis, infected cells were treated with different apoptosis-inducing agents. Surprisingly, EBOV-infected cells remained sensitive to apoptosis induced by external stimuli. Neither receptor- nor mitochondrion-mediated apoptosis signaling was inhibited in EBOV infection. Although double-stranded RNA (dsRNA)-induced activation of protein kinase R (PKR) was blocked in EBOV-infected cells, induction of apoptosis mediated by dsRNA was not suppressed. When EBOV-infected cells were treated with dsRNA-dependent caspase recruiter (dsCARE), an antiviral protein that selectively induces apoptosis in cells containing dsRNA, virus titers were strongly reduced. These data show that the inability of EBOV to block apoptotic pathways may open up new strategies toward the development of antiviral therapeutics. PMID:23468487

  5. AICAR induces mitochondrial apoptosis in human osteosarcoma cells through an AMPK-dependent pathway.

    PubMed

    Morishita, Masayuki; Kawamoto, Teruya; Hara, Hitomi; Onishi, Yasuo; Ueha, Takeshi; Minoda, Masaya; Katayama, Etsuko; Takemori, Toshiyuki; Fukase, Naomasa; Kurosaka, Masahiro; Kuroda, Ryosuke; Akisue, Toshihiro

    2017-01-01

    The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) modulates cellular energy metabolism, and promotes mitochondrial proliferation and apoptosis. Previous studies have shown that AICAR has anticancer effects in various cancers, however the roles of AMPK and/or the effects of AICAR on osteosarcoma have not been reported. In the present study, we evaluated the effects of AICAR on tumor growth and mitochondrial apoptosis in human osteosarcoma both in vitro and in vivo. For in vitro experiments, two human osteosarcoma cell lines, MG63 and KHOS, were treated with AICAR, and the effects of AICAR on cell growth and mitochondrial apoptosis were assessed by WST assays, TUNEL staining, and immunoblot analyses. In vivo, human osteosarcoma-bearing mice were treated with AICAR, and the mitochondrial proliferation and apoptotic activity in treated tumors were assessed. In vitro experiments revealed that AICAR activated AMPK, inhibited cell growth, and induced mitochondrial apoptosis in both osteosarcoma cell lines. In vivo, AICAR significantly reduced osteosarcoma growth without apparent body weight loss and AICAR increased both mitochondrial proliferation and apoptotic activity in treated tumor tissues. AICAR showed anticancer effects in osteosarcoma cells through an AMPK-dependent peroxisome proliferator‑activated receptor-γ coactivator-1α (PGC-1α)/mitochondrial transcription factor A (TFAM)/mitochondrial pathway. The findings in this study strongly suggest that AICAR could be considered as a potent therapeutic agent for the treatment of human osteosarcoma.

  6. AICAR induces mitochondrial apoptosis in human osteosarcoma cells through an AMPK-dependent pathway

    PubMed Central

    Morishita, Masayuki; Kawamoto, Teruya; Hara, Hitomi; Onishi, Yasuo; Ueha, Takeshi; Minoda, Masaya; Katayama, Etsuko; Takemori, Toshiyuki; Fukase, Naomasa; Kurosaka, Masahiro; Kuroda, Ryosuke; Akisue, Toshihiro

    2017-01-01

    The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) modulates cellular energy metabolism, and promotes mitochondrial proliferation and apoptosis. Previous studies have shown that AICAR has anticancer effects in various cancers, however the roles of AMPK and/or the effects of AICAR on osteosarcoma have not been reported. In the present study, we evaluated the effects of AICAR on tumor growth and mitochondrial apoptosis in human osteosarcoma both in vitro and in vivo. For in vitro experiments, two human osteosarcoma cell lines, MG63 and KHOS, were treated with AICAR, and the effects of AICAR on cell growth and mitochondrial apoptosis were assessed by WST assays, TUNEL staining, and immunoblot analyses. In vivo, human osteosarcoma-bearing mice were treated with AICAR, and the mitochondrial proliferation and apoptotic activity in treated tumors were assessed. In vitro experiments revealed that AICAR activated AMPK, inhibited cell growth, and induced mitochondrial apoptosis in both osteosarcoma cell lines. In vivo, AICAR significantly reduced osteosarcoma growth without apparent body weight loss and AICAR increased both mitochondrial proliferation and apoptotic activity in treated tumor tissues. AICAR showed anticancer effects in osteosarcoma cells through an AMPK-dependent peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)/mitochondrial transcription factor A (TFAM)/mitochondrial pathway. The findings in this study strongly suggest that AICAR could be considered as a potent therapeutic agent for the treatment of human osteosarcoma. PMID:27878239

  7. Mcl-1 involvement in mitochondrial dynamics is associated with apoptotic cell death

    PubMed Central

    Morciano, Giampaolo; Giorgi, Carlotta; Balestra, Dario; Marchi, Saverio; Perrone, Daniela; Pinotti, Mirko; Pinton, Paolo

    2016-01-01

    The B-cell lymphoma-2 (Bcl-2) family proteins are critical regulators of apoptosis and consist of both proapoptotic and antiapoptotic factors. Within this family, the myeloid cell leukemia factor 1 (Mcl-1) protein exists in two forms as the result of alternative splicing. The long variant (Mcl-1L) acts as an antiapoptotic factor, whereas the short isoform (Mcl-1S) displays proapoptotic activity. In this study, using splice-switching antisense oligonucleotides (ASOs), we increased the synthesis of Mcl-1S, which induced a concurrent reduction of Mcl-1L, resulting in increased sensitivity of cancer cells to apoptotic stimuli. The Mcl-1 ASOs also induced mitochondrial hyperpolarization and a consequent increase in mitochondrial calcium (Ca2+) accumulation. The high Mcl-1S/L ratio correlated with significant hyperfusion of the entire mitochondrial network, which occurred in a dynamin-related protein (Drp1)–dependent manner. Our data indicate that the balance between the long and short variants of the Mcl-1 gene represents a key aspect of the regulation of mitochondrial physiology. We propose that the Mcl-1L/S balance is a novel regulatory factor controlling the mitochondrial fusion and fission machinery. PMID:26538029

  8. Evidence that glucocorticoid- and cyclic AMP-induced apoptotic pathways in lymphocytes share distal events.

    PubMed Central

    Dowd, D R; Miesfeld, R L

    1992-01-01

    WEHI7.2 murine lymphocytes undergo apoptotic death when exposed to glucocorticoids or elevated levels of intracellular cyclic AMP (cAMP), and these pathways are initiated by the glucocorticoid receptor (GR) and protein kinase A, respectively. We report the isolation and characterization of a novel WEHI7.2 variant cell line, WR256, which was selected in a single step for growth in the presence of dexamethasone and arose at a frequency of approximately 10(-10). The defect was not GR-related, as WR256 expressed functional GR and underwent GR-dependent events associated with apoptosis, such as hormone-dependent gene transcription and inhibition of cell proliferation. Moreover, the glucocorticoid-resistant phenotype was stable in culture and did not revert after treatment with 5-azacytidine or upon stable expression of GR cDNA. In addition, WR256 did not exhibit the diminished mitochondrial activity commonly associated with apoptosis. Interestingly, WR256 was also found to be resistant to 8-bromo-cAMP and forskolin despite having normal levels of protein kinase A activity and the ability to induce cAMP-dependent transcription. We examined the steady-state transcript levels of bcl-2, a gene whose protein product acts dominantly to inhibit thymocyte apoptosis, to determine whether elevated bcl-2 expression could account for the resistant phenotype. Our data showed that bcl-2 RNA levels were similar in the two cell lines and not altered by either dexamethasone or 8-bromo-cAMP treatment. These results suggest that WR256 exhibits a "deathless" phenotype and has a unique defect in a step of the apoptotic cascade that may be common to the glucocorticoid- and cAMP-mediated cell death pathways. Images PMID:1378529

  9. Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells

    SciTech Connect

    Afeseh Ngwa, Hilary; Kanthasamy, Arthi; Gu, Yan; Fang, Ning; Anantharam, Vellareddy; Kanthasamy, Anumantha G.

    2011-11-15

    The production of man-made nanoparticles for various modern applications has increased exponentially in recent years, but the potential health effects of most nanoparticles are not well characterized. Unfortunately, in vitro nanoparticle toxicity studies are extremely limited by yet unresolved problems relating to dosimetry. In the present study, we systematically characterized manganese (Mn) nanoparticle sizes and examined the nanoparticle-induced oxidative signaling in dopaminergic neuronal cells. Differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) studies revealed that Mn nanoparticles range in size from single nanoparticles ({approx} 25 nM) to larger agglomerates when in treatment media. Manganese nanoparticles were effectively internalized in N27 dopaminergic neuronal cells, and they induced a time-dependent upregulation of the transporter protein transferrin. Exposure to 25-400 {mu}g/mL Mn nanoparticles induced cell death in a time- and dose-dependent manner. Mn nanoparticles also significantly increased ROS, accompanied by a caspase-mediated proteolytic cleavage of proapoptotic protein kinase C{delta} (PKC{delta}), as well as activation loop phosphorylation. Blocking Mn nanoparticle-induced ROS failed to protect against the neurotoxic effects, suggesting the involvement of other pathways. Further mechanistic studies revealed changes in Beclin 1 and LC3, indicating that Mn nanoparticles induce autophagy. Primary mesencephalic neuron exposure to Mn nanoparticles induced loss of TH positive dopaminergic neurons and neuronal processes. Collectively, our results suggest that Mn nanoparticles effectively enter dopaminergic neuronal cells and exert neurotoxic effects by activating an apoptotic signaling pathway and autophagy, emphasizing the need for assessing possible health risks associated with an increased use of Mn nanoparticles in modern applications. -- Highlights: Black-Right-Pointing-Pointer Mn nanoparticles

  10. Exogenous H2S regulates endoplasmic reticulum-mitochondria cross-talk to inhibit apoptotic pathways in STZ-induced type I diabetes.

    PubMed

    Yang, Fan; Yu, Xiangjing; Li, Ting; Wu, Jianjun; Zhao, Yajun; Liu, Jiaqi; Sun, Aili; Dong, Shiyun; Wu, Jichao; Zhong, Xin; Xu, Changqing; Lu, Fanghao; Zhang, Weihua

    2017-03-01

    The upregulation of reactive oxygen species (ROS) is a primary cause of cardiomyocyte apoptosis in diabetes cardiomyopathy (DCM). Mitofusin-2 (Mfn-2) is a key protein that bridges the mitochondria and endoplasmic reticulum (ER). Hydrogen sulfide (H2S)-mediated cardioprotection is related to antioxidant effects. The present study demonstrated that H2S inhibited the interaction between the ER and mitochondrial apoptotic pathway. This study investigated cardiac function, ultrastructural changes in the ER and mitochondria, apoptotic rate using TUNEL, and the expression of ER stress-associated proteins and mitochondrial apoptotic proteins in cardiac tissues in STZ-induced type I diabetic rats treated with or without NaHS (donor of H2S). Mitochondria of cardiac tissues were isolated, and MPTP opening and cytochrome c (cyt C) and Mfn-2 expression were also detected. Our data showed that hyperglycemia decreased the cardiac function by ultrasound cardiogram, and the administration of exogenous H2S ameliorated these changes. We demonstrated that the expression of ER stress sensors and apoptotic rates were elevated in cardiac tissue of DCM and cultured H9C2 cells, but the expression of these proteins was reduced following exogenous H2S treatment. The expression of mitochondrial apoptotic proteins, cyt C, and mPTP opening was decreased following treatment with exogenous H2S. In our experiment, the expression and immunofluorescence of Mfn-2 were both decreased after transfection with Mfn-2-siRNA. Hyperglycemia stimulated ER interactions and mitochondrial apoptotic pathways, which were inhibited by exogenous H2S treatment through the regulation of Mfn-2 expression.

  11. Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1

    PubMed Central

    Fond, Aaron M.; Lee, Chang Sup; Schulman, Ira G.; Kiss, Robert S.; Ravichandran, Kodi S.

    2015-01-01

    Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane–initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell–initiated pathway functioned independently of the liver X receptor (LXR) sterol–sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo. PMID:26075824

  12. 3-Nitropropionic acid induces autophagy by forming mitochondrial permeability transition pores rather than activatiing the mitochondrial fission pathway

    PubMed Central

    Solesio, Maria E; Saez-Atienzar, Sara; Jordan, Joaquin; Galindo, Maria F

    2013-01-01

    BACKGROUND AND PURPOSE Huntington's disease is a neurodegenerative process associated with mitochondrial alterations. Inhibitors of the electron–transport channel complex II, such as 3-nitropropionic acid (3NP), are used to study the molecular and cellular pathways involved in this disease. We studied the effect of 3NP on mitochondrial morphology and its involvement in macrophagy. EXPERIMENTAL APPROACH Pharmacological and biochemical methods were used to characterize the effects of 3NP on autophagy and mitochondrial morphology. SH-SY5Y cells were transfected with GFP-LC3, GFP-Drp1 or GFP-Bax to ascertain their role and intracellular localization after 3NP treatment using confocal microscopy. KEY RESULTS Untreated SH-SY5Y cells presented a long, tubular and filamentous net of mitochondria. After 3NP (5 mM) treatment, mitochondria became shorter and rounder. 3NP induced formation of mitochondrial permeability transition pores, both in cell cultures and in isolated liver mitochondria, and this process was inhibited by cyclosporin A. Participation of the mitochondrial fission pathway was excluded because 3NP did not induce translocation of the dynamin-related protein 1 (Drp1) to the mitochondria. The Drp1 inhibitor Mdivi-1 did not affect the observed changes in mitochondrial morphology. Finally, scavengers of reactive oxygen species failed to prevent mitochondrial alterations, while cyclosporin A, but not Mdivi-1, prevented the generation of ROS. CONCLUSIONS AND IMPLICATIONS There was a direct correlation between formation of mitochondrial permeability transition pores and autophagy induced by 3NP treatment. Activation of autophagy preceded the apoptotic process and was mediated, at least partly, by formation of reactive oxygen species and mitochondrial permeability transition pores. LINKED ARTICLE This article is commented on by González-Polo et al., pp. 60–62 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02203.x PMID

  13. Mitochondrial Targeting of Vitamin E Succinate Enhances Its Pro-apoptotic and Anti-cancer Activity via Mitochondrial Complex II*

    PubMed Central

    Dong, Lan-Feng; Jameson, Victoria J. A.; Tilly, David; Cerny, Jiri; Mahdavian, Elahe; Marín-Hernández, Alvaro; Hernández-Esquivel, Luz; Rodríguez-Enríquez, Sara; Stursa, Jan; Witting, Paul K.; Stantic, Bela; Rohlena, Jakub; Truksa, Jaroslav; Kluckova, Katarina; Dyason, Jeffrey C.; Ledvina, Miroslav; Salvatore, Brian A.; Moreno-Sánchez, Rafael; Coster, Mark J.; Ralph, Stephen J.; Smith, Robin A. J.; Neuzil, Jiri

    2011-01-01

    Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC50 of 80 μm, whereas the electron transfer from CII to CIII was inhibited with IC50 of 1.5 μm. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser68 within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug. PMID:21059645

  14. Mitochondrial targeting of vitamin E succinate enhances its pro-apoptotic and anti-cancer activity via mitochondrial complex II.

    PubMed

    Dong, Lan-Feng; Jameson, Victoria J A; Tilly, David; Cerny, Jiri; Mahdavian, Elahe; Marín-Hernández, Alvaro; Hernández-Esquivel, Luz; Rodríguez-Enríquez, Sara; Stursa, Jan; Witting, Paul K; Stantic, Bela; Rohlena, Jakub; Truksa, Jaroslav; Kluckova, Katarina; Dyason, Jeffrey C; Ledvina, Miroslav; Salvatore, Brian A; Moreno-Sánchez, Rafael; Coster, Mark J; Ralph, Stephen J; Smith, Robin A J; Neuzil, Jiri

    2011-02-04

    Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC(50) of 80 μM, whereas the electron transfer from CII to CIII was inhibited with IC(50) of 1.5 μM. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser(68) within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug.

  15. Physical exercise improves brain cortex and cerebellum mitochondrial bioenergetics and alters apoptotic, dynamic and auto(mito)phagy markers.

    PubMed

    Marques-Aleixo, I; Santos-Alves, E; Balça, M M; Rizo-Roca, D; Moreira, P I; Oliveira, P J; Magalhães, J; Ascensão, A

    2015-08-20

    We here investigate the effects of two exercise modalities (endurance treadmill training-TM and voluntary free-wheel activity-FW) on the brain cortex and cerebellum mitochondrial bioenergetics, permeability transition pore (mPTP), oxidative stress, as well as on proteins involved in mitochondrial biogenesis, apoptosis, and quality control. Eighteen male rats were assigned to sedentary-SED, TM and FW groups. Behavioral alterations and ex vivo brain mitochondrial function endpoints were assessed. Proteins involved in oxidative phosphorylation (OXPHOS, including the adenine nucleotide translocator), oxidative stress markers and regulatory proteins (SIRT3, p66shc, UCP2, carbonyls, MDA, -SH, aconitase, Mn-SOD), as well as proteins involved in mitochondrial biogenesis (PGC1α, TFAM) were evaluated. Apoptotic signaling was measured through quantifying caspase 3, 8 and 9-like activities, Bax, Bcl2, CypD, and cofilin expression. Mitochondrial dynamics (Mfn1/2, OPA1 and DRP1) and auto(mito)phagy (LC3II, Beclin1, Pink1, Parkin, p62)-related proteins were also measured by Western blotting. Only the TM exercise group showed increased spontaneous alternation and exploratory activity. Both exercise regimens improved mitochondrial respiratory activity, increased OXPHOS complexes I, III and V subunits in both brain subareas and decreased oxidative stress markers. Increased resistance to mPTP and decreased apoptotic signaling were observed in the brain cortex from TM and in the cerebellum from TM and FW groups. Also, exercise increased the expression of proteins involved in mitochondrial biogenesis, autophagy and fusion, simultaneous with decreased expression of mitochondrial fission-related protein DRP1. In conclusion, physical exercise improves brain cortex and cerebellum mitochondrial function, decreasing oxidative stress and apoptotic related markers. It is also possible that favorable alterations in mitochondrial biogenesis, dynamics and autophagy signaling induced by exercise

  16. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore

    PubMed Central

    Mandal, Tirtha; Shin, Seungjin; Aluvila, Sreevidya; Chen, Hui-Chen; Grieve, Carter; Choe, Jun-Yong; Cheng, Emily H.; Hustedt, Eric J.; Oh, Kyoung Joon

    2016-01-01

    In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the ‘α3/α5’ interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known ‘α6:α6’ interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH. PMID:27488021

  17. Activation of mitochondria-mediated apoptotic pathway in tri-ortho-cresyl phosphate-induced delayed neuropathy.

    PubMed

    Zou, Chaoshuang; Kou, Ruirui; Gao, Yuan; Xie, Keqin; Song, Fuyong

    2013-06-01

    Previous studies suggest that abnormal neurons death has been implicated in organophosphate-induced delayed neuropathy (OPIDN). However, the precise mechanism of neuronal death in OPIDN remains largely unknown. In this study, adult hens were treated with a dosage of 750 mg/kg tri-ortho-cresyl phosphate (TOCP) by gavage, and then sacrificed on the time-points of 1, 5, 10, and 21 days after dosing TOCP, respectively. The apoptotic change of spinal cord neurons induced by TOCP was examined, and the role of mitochondria-mediated apoptosis of neurons during OPIDN was investigated. TUNEL assays showed that apoptotic neurons in hen spinal cords began to appear on day 5 following TOCP exposure. Immunohistochemistry and western blot analysis revealed a translocation of cytochrome C from mitochondria to cytoplasm after dosing TOCP. Moreover, the level of Bcl-2, Bcl-xl, Pro-caspase3 and Pro-caspase9 in hen spinal cord was significantly decreased, whereas that of Bax and cleaved-PARP was significantly elevated. Taken together, these findings indicate that the administration of TOCP can induce neuron apoptosis in hen spinal cords, which might be mediated by the activation of mitochondrial apoptotic pathway.

  18. Mitochondrial apoptosis is dispensable for NLRP3 inflammasome activation but non-apoptotic caspase-8 is required for inflammasome priming

    PubMed Central

    Allam, Ramanjaneyulu; Lawlor, Kate E; Yu, Eric Chi-Wang; Mildenhall, Alison L; Moujalled, Donia M; Lewis, Rowena S; Ke, Francine; Mason, Kylie D; White, Michael J; Stacey, Katryn J; Strasser, Andreas; O’Reilly, Lorraine A; Alexander, Warren; Kile, Benjamin T; Vaux, David L; Vince, James E

    2014-01-01

    A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels. PMID:24990442

  19. Mitochondrial apoptosis is dispensable for NLRP3 inflammasome activation but non-apoptotic caspase-8 is required for inflammasome priming.

    PubMed

    Allam, Ramanjaneyulu; Lawlor, Kate E; Yu, Eric Chi-Wang; Mildenhall, Alison L; Moujalled, Donia M; Lewis, Rowena S; Ke, Francine; Mason, Kylie D; White, Michael J; Stacey, Katryn J; Strasser, Andreas; O'Reilly, Lorraine A; Alexander, Warren; Kile, Benjamin T; Vaux, David L; Vince, James E

    2014-09-01

    A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.

  20. Isorhamnetin inhibits H₂O₂-induced activation of the intrinsic apoptotic pathway in H9c2 cardiomyocytes through scavenging reactive oxygen species and ERK inactivation.

    PubMed

    Sun, Bing; Sun, Gui-Bo; Xiao, Jing; Chen, Rong-Chang; Wang, Xin; Wu, Ying; Cao, Li; Yang, Zhi-Hong; Sun, Xiao-Bo

    2012-02-01

    As a traditional Chinese medicine, the sea buckthorn (Hippophae rhamnoides L.) has a long history in the treatment of ischemic heart disease and circulatory disorders. However, the active compounds responsible for and the underlying mechanisms of these effects are not fully understood. In this article, isorhamnetin pretreatment counteracted H(2)O(2)-induced apoptotic damage in H9c2 cardiomyocytes. Isorhamnetin did not inhibit the death receptor-dependent or extrinsic apoptotic pathways, as characterized by its absence in both caspase-8 inactivation and tBid downregulation along with unchanged Fas and TNFR1 mRNA levels. Instead, isorhamnetin specifically suppressed the mitochondria-dependent or intrinsic apoptotic pathways, as characterized by inactivation of caspase-9 and -3, maintenance of the mitochondrial membrane potential (ΔΨm), and regulation of a series of Bcl-2 family genes upstream of ΔΨm. The anti-apoptotic effects of isorhamnetin were linked to decreased ROS generation. H(2)O(2) activated ERK and p53, whereas isorhamnetin inhibited their activation. ERK overexpression overrode the isorhamnetin-induced inhibition of the intrinsic apoptotic pathway in H9c2 cardiomyocytes, which indicated that an ERK-dependent pathway was involved. Furthermore, N-acetyl cysteine (a potent ROS scavenger) could attenuate the H(2)O(2)-induced apoptosis. However, PD98059 (an ERK-specific inhibitor) could not effectively antagonize ROS generation, which indicates that ROS may be an upstream inducer of ERK. In conclusion, isorhamnetin inhibits the H(2)O(2)-induced activation of the intrinsic apoptotic pathway via ROS scavenging and ERK inactivation. Therefore, isorhamnetin is a promising reagent for the treatment of ROS-induced cardiomyopathy.

  1. Apoptotic pathway induced by noscapine in human myelogenous leukemic cells.

    PubMed

    Heidari, Nastaran; Goliaei, Bahram; Moghaddam, Parvaneh Rahimi; Rahbar-Roshandel, Nahid; Mahmoudian, Massoud

    2007-11-01

    It has been shown that noscapine, an opium-derived phthalideisoquinoline alkaloid that is currently being used as an oral antitussive drug, induces apoptosis in myeloid leukemia cells. The molecular mechanism responsible for the anticancer effects of noscapine is poorly understood. In the current study, the apoptotic effects of noscapine on two myeloid cell lines, apoptosis-proficient HL60 cells and apoptosis-resistant K562 cells, were analyzed. An increase in the activity of caspase-2, -3, -6, -8 and -9, poly(ADP ribose) polymerase cleavage, detection of phosphatidylserine on the outer layer of the cell membrane, nucleation of chromatin, and DNA fragmentation suggested the induction of apoptosis. Noscapine increased the Bax/Bcl-2 ratio with a significant decrease of Bcl-2 expression accompanied with Bcl-2 phosphorylation. Using an inhibitory approach, the activation of the caspase cascade involved in the noscapine-induced apoptosis was analyzed. We observed no inhibitory effect of the caspase-8 inhibitor on caspase-9 activity. In view of these results and taking into consideration that K562 cells are Fas-null, we suggested that caspase-8 is activated in a Fas-independent manner downstream of caspase-9. In conclusion, noscapine can induce apoptosis in both apoptosis-proficient and apoptosis-resistant leukemic cells, and it can be a novel candidate in the treatment of hematological malignancies.

  2. Plasma-activated medium induces A549 cell injury via a spiral apoptotic cascade involving the mitochondrial-nuclear network.

    PubMed

    Adachi, Tetsuo; Tanaka, Hiromasa; Nonomura, Saho; Hara, Hirokazu; Kondo, Shin-ichi; Hori, Masaru

    2015-02-01

    Plasma medicine is a rapidly expanding new field of interdisciplinary research that combines physics, chemistry, biology, and medicine. Nonthermal atmospheric pressure plasma can be applied to living cells and tissues and has emerged as a novel technology for cancer therapy. Plasma has recently been shown to affect cells not only directly, but also by indirect treatment with previously prepared plasma-activated medium (PAM). The objective of this study was to demonstrate the inhibitory effects of PAM on A549 cell survival and elucidate the signaling mechanisms responsible for cell death. PAM maintained its ability to suppress cell viability for at least 1 week when stored at -80°C. The severity of PAM-triggered cell injury depended on the kind of culture medium used to prepare the PAM, especially that with or without pyruvate. Hydrogen peroxide (H2O2) and/or its derived or cooperating reactive oxygen species reduced the mitochondrial membrane potential, downregulated the expression of the antiapoptotic protein Bcl2, activated poly(ADP-ribose) polymerase-1, and released apoptosis-inducing factor from mitochondria with endoplasmic reticulum stress. However, the activation of caspase 3/7 and attenuation of cell viability by the addition of caspase inhibitor were not observed. The accumulation of adenine 5'-diphosphoribose as a product of the above reactions activated transient receptor potential melastatin 2, which elevated intracellular Ca(2+) levels and subsequently led to cell death. These results demonstrated that H2O2 and/or other reactive species in PAM disturbed the mitochondrial-nuclear network in cancer cells through a caspase-independent apoptotic pathway. Moreover, damage to the plasma membrane by H2O2-cooperating charged species not only induced apoptosis, but also increased its permeability to extracellular reactive species. These phenomena were also detected in PAM-treated HepG2 and MCF-7 cells.

  3. Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons

    PubMed Central

    Le, Shoshona S.; Loucks, F. Alexandra; Udo, Hiroshi; Richardson-Burns, Sarah; Phelps, Reid A.; Bouchard, Ron J.; Barth, Holger; Aktories, Klaus; Tyler, Kenneth L.; Kandel, Eric R.; Heidenreich, Kim A.; Linseman, Daniel A.

    2008-01-01

    Rho GTPases are key transducers of integrin/extracellular matrix and growth factor signaling. Although integrin-mediated adhesion and trophic support suppress neuronal apoptosis, the role of Rho GTPases in neuronal survival is unclear. Here, we have identified Rac as a critical pro-survival GTPase in cerebellar granule neurons (CGNs) and elucidated a death pathway triggered by its inactivation. GTP-loading of Rac1 was maintained in CGNs by integrin-mediated (RGDdependent) cell attachment and trophic support. Clostridium difficile toxin B (ToxB), a specific Rho family inhibitor, induced a selective caspase-mediated degradation of Rac1 without affecting RhoA or Cdc42 protein levels. Both ToxB and dominant-negative N17Rac1 elicited CGN apoptosis, characterized by cytochrome c release and activation of caspase-9 and -3, whereas dominant-negative N19RhoA or N17Cdc42 did not cause significant cell death. ToxB stimulated mitochondrial translocation and conformational activation of Bax, c-Jun activation, and induction of the BH3-only protein Bim. Similarly, c-Jun activation and Bim induction were observed with N17Rac1. A c-jun N-terminal protein kinase (JNK)/p38 inhibitor, SB203580, and a JNK-specific inhibitor, SP600125, significantly decreased ToxB-induced Bim expression and blunted each subsequent step of the apoptotic cascade. These results indicate that Rac acts downstream of integrins and growth factors to promote neuronal survival by repressing c-Jun/Bim-mediated mitochondrial apoptosis. PMID:16092944

  4. P38 MAP kinase mediates apoptosis after genipin treatment in non-small-cell lung cancer H1299 cells via a mitochondrial apoptotic cascade.

    PubMed

    Yang, Xue; Yao, Jie; Luo, Yue; Han, Yongguang; Wang, Zuobai; Du, Linfang

    2013-01-01

    Genipin, an active constituent of Gardenia fruit, has been reported to show an anti-tumor effect in several cancer cell systems. Here, we demonstrate how genipin exhibits a strong apoptotic cell death effect in human non-small-cell lung cancer H1299 cells. Genipin-mediated decrease in cell viability was observed through apoptosis as demonstrated by induction of a sub-G1 peak through flow cytometry, DNA fragmentation measured by TUNEL assay, and cleavage of poly ADP-ribose-polymerase. During genipin-induced apoptosis, the mitochondrial execution pathway was activated by caspase-9 and -3 activation as examined by a kinetic study, cytochrome c release, and a dose-dependent increase in Bax/Bcl-2 ratio. A search for the downstream pathway reveals that genipin-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2. SB203580, a p38MAPK inhibitor, markedly blocked the formation of TUNEL-positive apoptotic cells in genipin-treated cells. Besides, the interference of p38MAPK inhibited Bax expression and cytochrome c release. Altogether, our observations imply that genipin causes increased levels of Bax in response to p38MAPK signaling, which results in the initiation of mitochondrial death cascade, and therefore it holds promise as a potential chemotherapeutic agent for the treatment of H1299 cells.

  5. Activation of endoplasmic reticulum stress and the extrinsic apoptotic pathway in human lung cancer cells by the new synthetic flavonoid, LZ-205

    PubMed Central

    Li, Wei; Miao, Hanchi; Huang, Shaoliang; Zhou, Yuxin; Sun, Yang; Li, Zhiyu; Guo, Qinglong; Zhao, Li

    2016-01-01

    It has been shown that flavonoids have anti-tumor activity. In this study, LZ-205, a newly synthesized flavonoid, was found to be effective in inducing apoptosis in human lung cancer cells in vivo and in vitro. Mechanistically, LZ-205 triggers reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress and unfolded protein response, which could be reversed by silencing CHOP, a mediator of the ER stress-associated apoptosis. In addition, LZ-205-induced apoptosis is accompanied by the activation of both the mitochondrial apoptotic and extrinsic pathways, followed by decreased mitochondrial membrane potential (ΔΨm) and the alteration of the expression of mitochondria-related pro- and anti-apoptotic proteins. LZ-205 exhibits a potential antitumor effect in BALB/c nude mice bearing H460 tumor with low systemic toxicity. In summary, both the ROS-mediated ER stress pathway and the exogenous apoptotic pathway are involved in LZ-205-induced apoptosis in vitro and in vivo. Our data show a therapeutic potential of LZ-205 for the treatment of lung cancer. PMID:27895312

  6. Apoptosis and apoptotic pathway in actinic prurigo by immunohistochemistry

    PubMed Central

    Cuevas-González, Juan-Carlos; García-Vázquez, Francisco-Javier; Rodríguez-Lobato, Erika; Farfán-Morales, José-Eduardo

    2016-01-01

    Background Actinic prurigo (AP) is an idiopathic photodermatosis, this entity requires exposure to UV-B and -A to develop lesions. Apoptosis is a physiological death program that can be initiated by a permanently active mechanism (extrinsic pathway) or irreparable damage (intrinsic pathway). Material and Methods Descriptive study, the sample size comprised 64 paraffin blocks of tissue with a diagnosis of AP. In H&E-stained slides, the diagnosis of AP was corroborated, and 1-µm-thick sections were processed for immunohistochemistry (IHC). A database was constructed with SPSS version 20, Inc., Chicago, IL, USA, and descriptive statistics were analyzed by X2 test and comparison of means. Results A total of 64 cases were processed, of which 40 (62.5%) were cheilitis AP and 24 (37.5%) were AP in the skin. Of the 40 cheilitis samples, 27 were positive for Bcl-2 and caspase 3 (67.5%), p53 was expressed in 30 (75%). Of the skin lesions,p53 and caspase 3 were expressed in 18 of 24 cases (75%), and 13 were positive for Bcl-2 (54%). Conclusions We propose that apoptosis is the last step in the type IV subtype a-b hypersensitivity response-activation of the intrinsic pathway indicates that external factors, such as UV-A and -B are the trigger. Key words:Apoptosis, actinic prurigo, cheilitis actinic prurigo. PMID:26615506

  7. Reduction of apoptosis through the mitochondrial pathway by the administration of acetyl-L-carnitine to mouse fibroblasts in culture

    SciTech Connect

    Pillich, Rudolf Tito; Scarsella, Gianfranco; Risuleo, Gianfranco . E-mail: gianfranco.risuleo@uniroma1.it

    2005-05-15

    It is shown in literature that stress, such as deprivation of trophic factors and hypoxia, induces apoptosis in cultured cells and in tissues. In light of these results, we explored the possibility of protecting cells from programmed death by improving the metabolism of the mitochondrion. To this end, acetyl-L-carnitine was administered at various concentrations under conditions of serum deprivation. The choice of this drug was based on the accepted notion that acetyl-L-carnitine is able to stabilize mitochondrial membranes and to increase the supply of energy to the organelle. The results presented here indicate that the drug protects cells from apoptotic death: this is demonstrated by a lower positivity to the TUNEL reaction and by a strong reduction of the apoptotic DNA ladder in serum-deprived cells. The involvement of the mitochondrial apoptotic pathway was assessed by cytochrome C release and immunoreactivity to caspase 3. Moreover, acetyl-L-carnitine stimulates cell proliferation.

  8. Unraveling Biochemical Pathways Affected by Mitochondrial Dysfunctions Using Metabolomic Approaches

    PubMed Central

    Demine, Stéphane; Reddy, Nagabushana; Renard, Patricia; Raes, Martine; Arnould, Thierry

    2014-01-01

    Mitochondrial dysfunction(s) (MDs) can be defined as alterations in the mitochondria, including mitochondrial uncoupling, mitochondrial depolarization, inhibition of the mitochondrial respiratory chain, mitochondrial network fragmentation, mitochondrial or nuclear DNA mutations and the mitochondrial accumulation of protein aggregates. All these MDs are known to alter the capacity of ATP production and are observed in several pathological states/diseases, including cancer, obesity, muscle and neurological disorders. The induction of MDs can also alter the secretion of several metabolites, reactive oxygen species production and modify several cell-signalling pathways to resolve the mitochondrial dysfunction or ultimately trigger cell death. Many metabolites, such as fatty acids and derived compounds, could be secreted into the blood stream by cells suffering from mitochondrial alterations. In this review, we summarize how a mitochondrial uncoupling can modify metabolites, the signalling pathways and transcription factors involved in this process. We describe how to identify the causes or consequences of mitochondrial dysfunction using metabolomics (liquid and gas chromatography associated with mass spectrometry analysis, NMR spectroscopy) in the obesity and insulin resistance thematic. PMID:25257998

  9. Critical roles of Rho-associated kinase in membrane blebbing and mitochondrial pathway of apoptosis caused by 1-butanol.

    PubMed

    Noritake, Kanako; Aki, Toshihiko; Funakoshi, Takeshi; Unuma, Kana; Nara, Akina; Kato, Chizuru; Uemura, Koichi

    2012-09-01

    Alcohols are widely used as industrial solvents and chemical intermediates but can cause serious damage to human health. Nevertheless, few studies have addressed the molecular mechanisms underlying the cytotoxicity of industrial alcohols, with the notable exception of ethanol. The goal of our current study is to elucidate the molecular mechanism of cytotoxicity caused by primary alcohols containing longer carbon chains than ethanol. We find that 1-butanol induces morphological changes in H9c2 cardiomyoblastoma including nuclear condensation and membrane blebbing, both of which are features of apoptotic response. Moreover, a decrease in the mitochondrial membrane potential, the cytosolic release of cytochrome c, and the activation of caspase 9 and 3 was observed, thus revealing the activation of the mitochondrial apoptotic pathway by 1-butanol. The addition of Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed the membrane blebbing and mitochondrial apoptotic pathway. In comparison z-VAD-fmk, a pan-caspase inhibitor, did not inhibit membrane blebbing but did prevent cell death following exposure to 1-butanol. These results indicate that mitochondrial pathway of apoptosis and membrane blebbing are parallel phenomena that occur downstream of ROCK. This kinase thus plays an essential role in 1-butanol cytotoxicity and subsequent cell death in H9c2 cells.

  10. Detoxification of Mitochondrial Oxidants and Apoptotic Signaling Are Facilitated by Thioredoxin-2 and Peroxiredoxin-3 during Hyperoxic Injury

    PubMed Central

    Forred, Benjamin J.; Daugaard, Darwin R.; Titus, Brianna K.; Wood, Ryan R.; Floen, Miranda J.; Booze, Michelle L.

    2017-01-01

    Mitochondria play a fundamental role in the regulation of cell death during accumulation of oxidants. High concentrations of atmospheric oxygen (hyperoxia), used clinically to treat tissue hypoxia in premature newborns, is known to elicit oxidative stress and mitochondrial injury to pulmonary epithelial cells. A consequence of oxidative stress in mitochondria is the accumulation of peroxides which are detoxified by the dedicated mitochondrial thioredoxin system. This system is comprised of the oxidoreductase activities of peroxiredoxin-3 (Prx3), thioredoxin-2 (Trx2), and thioredoxin reductase-2 (TrxR2). The goal of this study was to understand the role of the mitochondrial thioredoxin system and mitochondrial injuries during hyperoxic exposure. Flow analysis of the redox-sensitive, mitochondrial-specific fluorophore, MitoSOX, indicated increased levels of mitochondrial oxidant formation in human adenocarcinoma cells cultured in 95% oxygen. Increased expression of Trx2 and TrxR2 in response to hyperoxia were not attributable to changes in mitochondrial mass, suggesting that hyperoxic upregulation of mitochondrial thioredoxins prevents accumulation of oxidized Prx3. Mitochondrial oxidoreductase activities were modulated through pharmacological inhibition of TrxR2 with auranofin and genetically through shRNA knockdown of Trx2 and Prx3. Diminished Trx2 and Prx3 expression was associated with accumulation of mitochondrial superoxide; however, only shRNA knockdown of Trx2 increased susceptibility to hyperoxic cell death and increased phosphorylation of apoptosis signal-regulating kinase-1 (ASK1). In conclusion, the mitochondrial thioredoxin system regulates hyperoxic-mediated death of pulmonary epithelial cells through detoxification of oxidants and regulation of redox-dependent apoptotic signaling. PMID:28045936

  11. Antitumor effects of traditional Chinese medicine targeting the cellular apoptotic pathway

    PubMed Central

    Xu, Huanli; Zhao, Xin; Liu, Xiaohui; Xu, Pingxiang; Zhang, Keming; Lin, Xiukun

    2015-01-01

    Defects in apoptosis are common phenomena in many types of cancer and are also a critical step in tumorigenesis. Targeting the apoptotic pathway has been considered an intriguing strategy for cancer therapy. Traditional Chinese medicine (TCM) has been used in the People’s Republic of China for thousands of years, and many of the medicines have been confirmed to be effective in the treatment of a number of tumors. With increasing cancer rates worldwide, the antitumor effects of TCMs have attracted more and more attention globally. Many of the TCMs have been shown to have antitumor activity through multiple targets, and apoptosis pathway-related targets have been extensively studied and defined to be promising. This review focuses on several antitumor TCMs, especially those with clinical efficacy, based on their effects on the apoptotic signaling pathway. The problems with and prospects of development of TCMs as anticancer agents are also presented. PMID:26056434

  12. BAD-mediated apoptotic pathway is associated with human cancer development.

    PubMed

    Stickles, Xiaomang B; Marchion, Douglas C; Bicaku, Elona; Al Sawah, Entidhar; Abbasi, Forough; Xiong, Yin; Bou Zgheib, Nadim; Boac, Bernadette M; Orr, Brian C; Judson, Patricia L; Berry, Amy; Hakam, Ardeshir; Wenham, Robert M; Apte, Sachin M; Berglund, Anders E; Lancaster, Johnathan M

    2015-04-01

    The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, p<0.001), breast (n=185, p<0.0008; n=61, p=0.04), colon (n=22, p<0.001) and endometrial (n=33, p<0.001) cancers, as well as with ovarian endometriosis (n=20, p<0.001). Higher pBAD protein levels were observed in the cancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, p<0.001). The increased pBAD protein levels after the depletion of PP2C conferred a growth advantage to the immortalized normal and cancer cells. The BAD-mediated apoptotic pathway

  13. Interconnections between apoptotic, autophagic and necrotic pathways: implications for cancer therapy development.

    PubMed

    Jain, Mayur V; Paczulla, Anna M; Klonisch, Thomas; Dimgba, Florence N; Rao, Sahana B; Roberg, Karin; Schweizer, Frank; Lengerke, Claudia; Davoodpour, Padideh; Palicharla, Vivek R; Maddika, Subbareddy; Łos, Marek

    2013-01-01

    The rapid accumulation of knowledge on apoptosis regulation in the 1990s was followed by the development of several experimental anticancer- and anti-ischaemia (stroke or myocardial infarction) drugs. Activation of apoptotic pathways or the removal of cellular apoptotic inhibitors has been suggested to aid cancer therapy and the inhibition of apoptosis was thought to limit ischaemia-induced damage. However, initial clinical studies on apoptosis-modulating drugs led to unexpected results in different clinical conditions and this may have been due to co-effects on non-apoptotic interconnected cell death mechanisms and the 'yin-yang' role of autophagy in survival versus cell death. In this review, we extend the analysis of cell death beyond apoptosis. Upon introduction of molecular pathways governing autophagy and necrosis (also called necroptosis or programmed necrosis), we focus on the interconnected character of cell death signals and on the shared cell death processes involving mitochondria (e.g. mitophagy and mitoptosis) and molecular signals playing prominent roles in multiple pathways (e.g. Bcl2-family members and p53). We also briefly highlight stress-induced cell senescence that plays a role not only in organismal ageing but also offers the development of novel anticancer strategies. Finally, we briefly illustrate the interconnected character of cell death forms in clinical settings while discussing irradiation-induced mitotic catastrophe. The signalling pathways are discussed in their relation to cancer biology and treatment approaches.

  14. BID links ferroptosis to mitochondrial cell death pathways.

    PubMed

    Neitemeier, Sandra; Jelinek, Anja; Laino, Vincenzo; Hoffmann, Lena; Eisenbach, Ina; Eying, Roman; Ganjam, Goutham K; Dolga, Amalia M; Oppermann, Sina; Culmsee, Carsten

    2017-03-09

    Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the Xc(-) system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by Xc(-) inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death.

  15. Multiple pathways influence mitochondrial inheritance in budding yeast.

    PubMed

    Frederick, Rebecca L; Okamoto, Koji; Shaw, Janet M

    2008-02-01

    Yeast mitochondria form a branched tubular network. Mitochondrial inheritance is tightly coupled with bud emergence, ensuring that daughter cells receive mitochondria from mother cells during division. Proteins reported to influence mitochondrial inheritance include the mitochondrial rho (Miro) GTPase Gem1p, Mmr1p, and Ypt11p. A synthetic genetic array (SGA) screen revealed interactions between gem1Delta and deletions of genes that affect mitochondrial function or inheritance, including mmr1Delta. Synthetic sickness of gem1Delta mmr1Delta double mutants correlated with defective mitochondrial inheritance by large buds. Additional studies demonstrated that GEM1, MMR1, and YPT11 each contribute to mitochondrial inheritance. Mitochondrial accumulation in buds caused by overexpression of either Mmr1p or Ypt11p did not depend on Gem1p, indicating these three proteins function independently. Physical linkage of mitochondria with the endoplasmic reticulum (ER) has led to speculation that distribution of these two organelles is coordinated. We show that yeast mitochondrial inheritance is not required for inheritance or spreading of cortical ER in the bud. Moreover, Ypt11p overexpression, but not Mmr1p overexpression, caused ER accumulation in the bud, revealing a potential role for Ypt11p in ER distribution. This study demonstrates that multiple pathways influence mitochondrial inheritance in yeast and that Miro GTPases have conserved roles in mitochondrial distribution.

  16. Exopolysaccharide from Trichoderma pseudokoningii induces the apoptosis of MCF-7 cells through an intrinsic mitochondrial pathway.

    PubMed

    Wang, Guodong; Liu, Chunyan; Liu, Jun; Liu, Bo; Li, Ping; Qin, Guozheng; Xu, Yanghui; Chen, Ke; Liu, Huixia; Chen, Kaoshan

    2016-01-20

    In this study, we reported the anticancer efficacy of exopolysaccharide (EPS) derived from Trichoderma pseudokoningii, on human breast cancer MCF-7 cells. Our results showed that EPS inhibited the proliferation of MCF-7 cells and induced lactic dehydrogenase release by inducing apoptosis and cell arrest at S phase. Further study revealed that EPS-induced apoptosis of MCF-7 cells was associated with alteration of nuclear morphology, disruption of mitochondrial membrane potential and accumulation of intracellular reactive oxygen species. Sequentially, EPS increased the activation of caspase-9 and caspase-3 in a dose-dependent manner; however, caspase-8 remained intact. Western blot analysis revealed that EPS increased the ratio of Bax/Bcl-2 and promoted the release of cytochrome c into the cytoplasm. Taken together, these findings provided evidence that EPS induced the apoptosis of MCF-7 cells through an intrinsic mitochondrial apoptotic pathway and that EPS may therefore be considered as an effective adjuvant agent against human breast cancer.

  17. Attenuation of Magnesium Sulfate on CoCl₂-Induced Cell Death by Activating ERK1/2/MAPK and Inhibiting HIF-1α via Mitochondrial Apoptotic Signaling Suppression in a Neuronal Cell Line.

    PubMed

    Huang, Chih-Yang; Hsieh, You-Liang; Ju, Da-Tong; Lin, Chien-Chung; Kuo, Chia-Hua; Liou, Yi-Fan; Ho, Tsung-Jung; Tsai, Chang-Hai; Tsai, Fuu-Jen; Lin, Jing-Ying

    2015-08-31

    Magnesium sulfate (MgSO₄) ameliorates hypoxia/ischemia-induced neuronal apoptosis in a rat model. This study aimed to investigate the mechanisms governing the anti-apoptotic effect of MgSO₄ on cobalt chloride (CoCl₂)-exposed NB41A3 mouse neuroblastoma cells. MgSO₄ increased the viability of NB41A3 cells treated with CoCl₂ in a dose-dependent manner. MgSO₄ treatment was shown to lead to an increase in the anti-apoptotic Bcl-2 family proteins, with a concomitant decrease in the pro-apoptotic proteins. MgSO₄ also attenuated the CoCl₂-induced disruption of mitochondrial membrane potential (ΔΨ(m)) and reduced the release of cytochrome c form the mitochondria to the cytosol. Furthermore, exposure to CoCl₂ caused activation of the hypoxia-inducible factor 1α (HIF-1α). On the other hand, MgSO₄ markedly reduced CoCl₂-induced HIF-1α activation and suppressed HIF-1α downstream protein BNIP3. MgSO₄ treatment induced ERK1/2 activation and attenuated CoCl₂-induced activation of p38 and JNK. Addition of the ERK1/2 inhibitor U0126 significantly reduced the ability of MgSO₄ to protect neurons from CoCl₂-induced mitochondrial apoptotic events. However, incubation of cultures with the p38 and JNK inhibitors did not significantly affect MgSO₄-mediated neuroprotection. MgSO₄ appears to suppress CoCl₂-induced NB41A3 cell death by activating ERK1/2/ MAPK pathways, which further modulates the role of Bcl-2 family proteins and mitochondria in NB41A3 cells. Our data suggest that MgSO₄ may act as a survival factor that preserves mitochondrial integrity and inhibits apoptotic pathways.

  18. An inhibitory mono-ubiquitylation of the Drosophila initiator caspase Dronc functions in both apoptotic and non-apoptotic pathways

    PubMed Central

    Ditzel, Mark; Meier, Pascal

    2017-01-01

    Apoptosis is an evolutionary conserved cell death mechanism, which requires activation of initiator and effector caspases. The Drosophila initiator caspase Dronc, the ortholog of mammalian Caspase-2 and Caspase-9, has an N-terminal CARD domain that recruits Dronc into the apoptosome for activation. In addition to its role in apoptosis, Dronc also has non-apoptotic functions such as compensatory proliferation. One mechanism to control the activation of Dronc is ubiquitylation. However, the mechanistic details of ubiquitylation of Dronc are less clear. For example, monomeric inactive Dronc is subject to non-degradative ubiquitylation in living cells, while ubiquitylation of active apoptosome-bound Dronc triggers its proteolytic degradation in apoptotic cells. Here, we examined the role of non-degradative ubiquitylation of Dronc in living cells in vivo, i.e. in the context of a multi-cellular organism. Our in vivo data suggest that in living cells Dronc is mono-ubiquitylated on Lys78 (K78) in its CARD domain. This ubiquitylation prevents activation of Dronc in the apoptosome and protects cells from apoptosis. Furthermore, K78 ubiquitylation plays an inhibitory role for non-apoptotic functions of Dronc. We provide evidence that not all of the non-apoptotic functions of Dronc require its catalytic activity. In conclusion, we demonstrate a mechanism whereby Dronc’s apoptotic and non-apoptotic activities can be kept silenced in a non-degradative manner through a single ubiquitylation event in living cells. PMID:28207763

  19. Natural terpenes prevent mitochondrial dysfunction, oxidative stress and release of apoptotic proteins during nimesulide-hepatotoxicity in rats.

    PubMed

    Singh, Brijesh Kumar; Tripathi, Madhulika; Chaudhari, Bhushan P; Pandey, Pramod K; Kakkar, Poonam

    2012-01-01

    Nimesulide, an anti-inflammatory and analgesic drug, is reported to cause severe hepatotoxicity. In this study, molecular mechanisms involved in deranged oxidant-antioxidant homeostasis and mitochondrial dysfunction during nimesulide-induced hepatotoxicity and its attenuation by plant derived terpenes, camphene and geraniol has been explored in male Sprague-Dawley rats. Hepatotoxicity due to nimesulide (80 mg/kg BW) was evident from elevated SGPT, SGOT, bilirubin and histo-pathological changes. Antioxidants and key redox enzymes (iNOS, mtNOS, Cu/Zn-SOD, Mn-SOD, GPx and GR) were altered significantly as assessed by their mRNA expression, Immunoblot analysis and enzyme activities. Redox imbalance along with oxidative stress was evident from decreased NAD(P)H and GSH (56% and 74% respectively; P<0.001), increased superoxide and secondary ROS/RNS generation along with oxidative damage to cellular macromolecules. Nimesulide reduced mitochondrial activity, depolarized mitochondria and caused membrane permeability transition (MPT) followed by release of apoptotic proteins (AIF; apoptosis inducing factor, EndoG; endonuclease G, and Cyto c; cytochrome c). It also significantly activated caspase-9 and caspase-3 and increased oxidative DNA damage (level of 8-Oxoguanine glycosylase; P<0.05). A combination of camphene and geraniol (CG; 1:1), when pre-administered in rats (10 mg/kg BW), accorded protection against nimesulide hepatotoxicity in vivo, as evident from normalized serum biomarkers and histopathology. mRNA expression and activity of key antioxidant and redox enzymes along with oxidative stress were also normalized due to CG pre-treatment. Downstream effects like decreased mitochondrial swelling, inhibition in release of apoptotic proteins, prevention of mitochondrial depolarization along with reduction in oxidized NAD(P)H and increased mitochondrial electron flow further supported protective action of selected terpenes against nimesulide toxicity. Therefore CG, a

  20. The cytotoxic and pro-apoptotic effects of phenylephrine on corneal stromal cells via a mitochondrion-dependent pathway both in vitro and in vivo.

    PubMed

    Zhao, Jun; Qiu, Yue; Tian, Cheng-Lei; Fan, Ting-Jun

    2016-08-01

    Phenylephrine (PHE), a selective α1-adrenergic receptor agonist, is often used as a decongestant for mydriasis prior to cataract surgery, and its abuse might be cytotoxic to the cornea and result in blurred vision. However, the cytotoxicity of PHE to the cornea and its cellular and molecular mechanisms remain unknown. To provide references for secure medication and prospective therapeutic interventions of PHE, we investigated the cytotoxicity of PHE to corneal stroma and its possible mechanisms using an in vitro model of human corneal stromal (HCS) cells and an in vivo model of cat keratocytes. We found that PHE, above the concentration of 0.0781125% (1/128 of its clinical therapeutic dosage), had a dose- and time-dependent cytotoxicity to HCS cells by inducing morphological abnormality and viability decline, as well as S phase arrest. Moreover, PHE induced apoptosis of HCS cells by inducing plasma membrane permeability elevation, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation. Furthermore, PHE could induce activations of caspase-3 and -9, disruption of mitochondrial transmembrane potential, downregulation of anti-apoptotic Bcl-xL, upregulation of pro-apoptotic Bax, along with upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. The cytotoxic and pro-apoptotic effects of PHE were also proven by the induced apoptotic-like ultrastructural alterations of keratocytes in vivo. Taken together, our results suggest that PHE has a significant cytotoxicity to corneal stroma cells both in vitro and in vivo by inducing cell apoptosis, and the pro-apoptotic effect of PHE is achieved via a Bcl-2 family proteins-mediated mitochondrion-dependent pathway.

  1. High fructose causes cardiac hypertrophy via mitochondrial signaling pathway

    PubMed Central

    Zhang, Yan-Bo; Meng, Yan-Hai; Chang, Shuo; Zhang, Rong-Yuan; Shi, Chen

    2016-01-01

    High fructose diet can cause cardiac hypertrophy and oxidative stress is a key mediator for myocardial hypertrophy. Disruption of cystic fibrosis transmembrane conductance regulator (CFTR) leads to oxidative stress. This study aims to reveal mitochondrial oxidative stress-related signaling pathway in high fructose-induced cardiac hypertrophy. Mice were fed high fructose to develop cardiac hypertrophy. Fructose and H2O2 were used to induce cardiomyocyte hypertrophy in vitro. Mitochondria-targeted antioxidant SkQ1 was applied to investigate the possible role of mitochondrial reactive oxygen species (ROS). CFTR silence was performed to detect the role of CFTR in high fructose-induced myocardial hypertrophy. ROS, glutathione (GSH), mitochondrial function and hypertrophic markers were measured. We confirmed that long-term high fructose diet caused cardiac hypertrophy and diastolic dysfunction and elevated mitochondrial ROS. However, SkQ1 administration prevented heart hypertrophy and mitochondrial oxidative stress. Cadiomyocytes incubated with fructose or H2O2 exhibited significantly increased cell areas but SkQ1 treatment ameliorated cardiomyocyte hypertrophy induced by high fructose or H2O2 in vitro. Those results revealed that the underlying mechanism for high fructose-induced heart hypertrophy was attributed to mitochondrial oxidative stress. Moreover, CFTR expression was decreased by high fructose intervention and CFTR silence resulted in an increase in mitochondrial ROS, which suggested high fructose diet affected mitochondrial oxidative stress by regulating CFTR expression. Electron transport chain impairment might be related to mitochondrial oxidative damage. In conclusion, our findings indicated that mitochondrial oxidative stress plays a central role in pathogenesis of high fructose-induced cardiac hypertrophy. High fructose decreases CFTR expression to regulate mitochondrial oxidative stress. PMID:27904687

  2. Flagellin/TLR5 responses in epithelia reveal intertwined activation of inflammatory and apoptotic pathways

    PubMed Central

    Zeng, Hui; Wu, Huixia; Sloane, Valerie; Jones, Rheinallt; Yu, Yimin; Lin, Patricia; Gewirtz, Andrew T.; Neish, Andrew S.

    2015-01-01

    Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we found that purified flagellin could recapitulate the human epithelial cell proinflammatory responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory activation showed similar kinetics and gene specificity as that induced by the classical endogenous proinflammatory cytokine TNF-α, although both responses were more rapid than that elicited by viable flagellated bacteria. Flagellin, like TNF-α, activated a number of antiapoptotic mediators, and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent bacterially mediated apoptotic challenge. However, when NF-κB-mediated or phosphatidylinositol 3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8) activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of flagellin with epithelial cells induces caspase activation in parallel with proinflammatory responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial products suggests roles for host programmed cell death in the pathogenesis of enteric infections. PMID:16179598

  3. Saikosaponin D acts against corticosterone-induced apoptosis via regulation of mitochondrial GR translocation and a GR-dependent pathway.

    PubMed

    Li, Zong-Yang; Jiang, Yu-Mao; Liu, Ya-Min; Guo, Zhi; Shen, Sheng-Nan; Liu, Xin-Min; Pan, Rui-Le

    2014-08-04

    Saikosaponin D is an agonist of the glucocorticoid receptor (GR), and our preliminary study showed that it possesses neuroprotective effects in corticosterone-treated PC12 cells. However, further proof is required, and the molecular mechanisms of this neuroprotection remain unclear. This study sought to further examine the cytoprotective efficiency and potential mechanisms of action of Saikosaponin D in corticosterone-treated PC12 cells. The cells were treated with 250 μM corticosterone in the absence or presence of Saikosaponin D for 24 h; cell viability was then determined, and Hoechst 33342/propidium iodide (PI) and annexin/PI double staining, and TUNEL staining were performed. Next, mPTP, MMP, [Ca(2+)]i, translocation of the GR to the nucleus and Western blot analyses for caspase-3, caspase-9, cytochrome C, GR, GILZ, SGK-1, NF-Κb (P65), IκB-α, Bad, Akt, Hsp90 and HDAC-6 were investigated. The neuroprotective effects of Saikosaponin D were further confirmed by Hoechst 33342/PI, annexin/PI and TUNEL staining assays. These additional data suggested that Saikosaponin D partially reversed the physiological changes induced by corticosterone by inhibiting the translocation of the GR to the mitochondria, restoring mitochondrial function, down-regulating the expression of pro-apoptotic-related signalling events and up-regulating anti-apoptotic-related signalling events. These findings suggest that SSD exhibited its anti-apoptotic effects via differential regulation of mitochondrial and nuclear GR translocation, partial reversal of mitochondrial dysfunction, inhibition of the mitochondrial apoptotic pathway, and selective activation of the GR-dependent survival pathway.

  4. Frequent truncating mutation of TFAM induces mitochondrial DNA depletion and apoptotic resistance in microsatellite-unstable colorectal cancer.

    PubMed

    Guo, Jianhui; Zheng, Li; Liu, Wenyong; Wang, Xianshu; Wang, Zemin; Wang, Zehua; French, Amy J; Kang, Dongchon; Chen, Lin; Thibodeau, Stephen N; Liu, Wanguo

    2011-04-15

    The mitochondrial transcription factor A (TFAM) is required for mitochondrial DNA (mtDNA) replication and transcription. Disruption of TFAM results in heart failure and premature aging in mice. But very little is known about the role of TFAM in cancer development. Here, we report the identification of frequent frameshift mutations in the coding mononucleotide repeat of TFAM in sporadic colorectal cancer (CRC) cell lines and in primary tumors with microsatellite instability (MSI), but not in microsatellite stable (MSS) CRC cell lines and tumors. The presence of the TFAM truncating mutation, in CRC cells with MSI, reduced the TFAM protein level in vivo and in vitro and correlated with mtDNA depletion. Furthermore, forced overexpression of wild-type TFAM in RKO cells carrying a TFAM truncating mutation suppressed cell proliferation and inhibited RKO cell-induced xenograft tumor growth. Moreover, these cells showed more susceptibility to cisplatin-induced apoptosis due to an increase of cytochrome b (Cyt b) expression and its release from mitochondria. An interaction assay between TFAM and the heavy-strand promoter (HSP) of mitochondria revealed that mutant TFAM exhibited reduced binding to HSP, leading to reduction in Cyt b transcription. Collectively, these data provide evidence that a high incidence of TFAM truncating mutations leads to mitochondrial copy number reduction and mitochondrial instability, distinguishing most CRC with MSI from MSS CRC. These mutations may play an important role in tumorigenesis and cisplatin-induced apoptotic resistance of most microsatellite-unstable CRCs.

  5. YCl3 Promotes Neuronal Cell Death by Inducing Apoptotic Pathways in Rats

    PubMed Central

    Ding, Yechun; Tian, Yuantong; Zeng, Zhaoyi; Shuai, Ping; Lan, Haiying; Zhu, Xianshen; Zhong, Yi

    2017-01-01

    The pollutants rare earth elements (REEs) have posed great threats to human health. To investigate the cytotoxicity of yttrium (Y), a model that rats have free access to water containing YCl3 for 6 months is utilized. The results showed that YCl3 treatment promoted neuronal cell apoptosis by upregulating the proapoptotic factors Bax, caspase-3, Cyto c, and DAPK and by downregulating the antiapoptotic factors Bcl-2 and XIAP at both mRNA and protein levels. Conclusively, YCl3 exhibited cytotoxicity and promoted neuronal cell death by the induction of apoptotic pathways. PMID:28326317

  6. Both intrinsic and extrinsic apoptotic pathways are involved in Toll-like receptor 4 (TLR4)-induced cell death in monocytic THP-1 cells.

    PubMed

    Liu, Bei; Sun, Ruili; Luo, Hongbo; Liu, Xueting; Jiang, Manli; Yuan, Chuang; Yang, Li; Hu, Jinyue

    2017-02-01

    Our previous study showed that TLR3 induces apoptosis via both death receptors and mitochondial in human endothelial cells. We report here that the activation of TLR4 induced dose- and time-dependent cell death in moncytic THP-1 cells. LPS treatment of THP-1 cells induced the activation of both caspase 8 and 9, suggesting the involvement of intrinsic and extrinsic apoptosis pathways. TNFα was induced by TLR4 activation at both mRNA and protein levels, but its neutralization did not down-regulated TLR4-induced cell death. TLR4 activation also induced the up-regulation of TRAIL and its receptors DR4 and DR5, and the neutralization of TRAIL ameliorated TLR4 induced apoptosis, suggesting the involvement of TRAIL and its receptors DR4 and DR5 in LPS-induced cell death. Meanwhile, LPS treatment down-regulated the expression of FLICE inhibitory protein (FLIP), a suppressor of death receptor-induced cell death. In addition, TLR4 activation down-regulated the anti-apoptotic protein bcl-2, and up-regulated the pro-apoptotic proteins Noxa and Puma, suggesting that mitochondrial apoptotic pathway was also involved in LPS-induced cell death. Furthermore, we found that TAP63α might confer to the activation of intrinsic and extrinsic apoptotic pathways. The treatment of THP-1 cells with LPS induced the translocation of TAP63α from cytoplasm to nucleus. Taken together, our study suggested that both death receptors and mitochondial were involved in TLR4-induced cell death, and TAP63α may be a target for the prevention of LPS-induced cell death.

  7. The intrinsic apoptosis pathway mediates the pro-longevity response to mitochondrial ROS in C. elegans.

    PubMed

    Yee, Callista; Yang, Wen; Hekimi, Siegfried

    2014-05-08

    The increased longevity of the C. elegans electron transport chain mutants isp-1 and nuo-6 is mediated by mitochondrial ROS (mtROS) signaling. Here we show that the mtROS signal is relayed by the conserved, mitochondria-associated, intrinsic apoptosis signaling pathway (CED-9/Bcl2, CED-4/Apaf1, and CED-3/Casp9) triggered by CED-13, an alternative BH3-only protein. Activation of the pathway by an elevation of mtROS does not affect apoptosis but protects from the consequences of mitochondrial dysfunction by triggering a unique pattern of gene expression that modulates stress sensitivity and promotes survival. In vertebrates, mtROS induce apoptosis through the intrinsic pathway to protect from severely damaged cells. Our observations in nematodes demonstrate that sensing of mtROS by the apoptotic pathway can, independently of apoptosis, elicit protective mechanisms that keep the organism alive under stressful conditions. This results in extended longevity when mtROS generation is inappropriately elevated. These findings clarify the relationships between mitochondria, ROS, apoptosis, and aging.

  8. Over-expression of mitochondrial ferritin affects the JAK2/STAT5 pathway in K562 cells and causes mitochondrial iron accumulation

    PubMed Central

    Santambrogio, Paolo; Erba, Benedetta Gaia; Campanella, Alessandro; Cozzi, Anna; Causarano, Vincenza; Cremonesi, Laura; Gallì, Anna; Della Porta, Matteo Giovanni; Invernizzi, Rosangela; Levi, Sonia

    2011-01-01

    Background Mitochondrial ferritin is a nuclear encoded iron-storage protein localized in mitochondria. It has anti-oxidant properties related to its ferroxidase activity, and it is able to sequester iron avidly into the organelle. The protein has a tissue-specific pattern of expression and is also highly expressed in sideroblasts of patients affected by hereditary sideroblastic anemia and by refractory anemia with ringed sideroblasts. The present study examined whether mitochondrial ferritin has a role in the pathogenesis of these diseases. Design and Methods We analyzed the effect of mitochondrial ferritin over-expression on the JAK2/STAT5 pathway, on iron metabolism and on heme synthesis in erythroleukemic cell lines. Furthermore its effect on apoptosis was evaluated on human erythroid progenitors. Results Data revealed that a high level of mitochondrial ferritin reduced reactive oxygen species and Stat5 phosphorylation while promoting mitochondrial iron loading and cytosolic iron starvation. The decline of Stat5 phosphorylation induced a decrease of the level of anti-apoptotic Bcl-xL transcript compared to that in control cells; however, transferrin receptor 1 transcript increased due to the activation of the iron responsive element/iron regulatory protein machinery. Also, high expression of mitochondrial ferritin increased apoptosis, limited heme synthesis and promoted the formation of Perls-positive granules, identified by electron microscopy as iron granules in mitochondria. Conclusions Our results provide evidence suggesting that Stat5-dependent transcriptional regulation is displaced by strong cytosolic iron starvation status induced by mitochondrial ferritin. The protein interferes with JAK2/STAT5 pathways and with the mechanism of mitochondrial iron accumulation. PMID:21712541

  9. Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

    SciTech Connect

    Igaki, Tatsushi; Suzuki, Yasuyuki; Tokushige, Naoko; Aonuma, Hiroka; Takahashi, Ryosuke . E-mail: ryosuket@kuhp.kyoto-u.ac.jp; Miura, Masayuki . E-mail: miura@mol.f.u-tokyo.ac.jp

    2007-05-18

    Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution.

  10. Arsenic induces apoptosis by the lysosomal-mitochondrial pathway in INS-1 cells.

    PubMed

    Pan, Xiao; Jiang, Liping; Zhong, Laifu; Geng, Chengyan; Jia, Li; Liu, Shuang; Guan, Huai; Yang, Guang; Yao, Xiaofeng; Piao, Fengyuan; Sun, Xiance

    2016-02-01

    Recently, long term arsenic exposure was considered to be associated with an increased risk of diabetes mellitus. While a relation of cause-and-effect between apoptosis of pancreatic β-cells and arsenic exposure, the precise mechanisms of these events remains unclear. The aim of this study was to explore arsenic-induced pancreatic β-cell apoptosis and the mechanisms of through the possible link between lysosomal and the mitochondrial apoptotic pathway. After exposure to 10 μM of arsenic, the reactive oxygen species (ROS) level was significantly increased at 12 h, while the mitochondrial membrane potential was reduced at 24 h and the lysosomal membrane integrity was disrupted at 48 h. A significant increase in protein expression for cytochrome c was also observed using Western blot analysis after exposure to arsenic for 48 h. To further demonstrate that arsenic reduced the lysosomal membrane integrity, cells pretreated with NH4 Cl and exposed to arsenic harbored a lower fluorescence increase than cells that were only exposed to arsenic. In addition, apoptosis was mesured using Hoechst 33342/PI dual staining by microscopy and annexin V-FITC/propidium iodide dual staining by flow cytometry. The results show an increased uptake of the arsenic dose and the cells changed from dark blue to light blue, karyopyknosis, nuclear chromatin condensation, side set or fracture, and a correlation was found between the number of apoptotic cells and arsenic dose. The result of present study suggest that arsenic may induce pancreatic β-cell apoptosis through activation of the lysosome-mitochondrial pathway.

  11. Potential of apoptotic pathway-targeted cancer therapeutic research: Where do we stand?

    PubMed

    Baig, S; Seevasant, I; Mohamad, J; Mukheem, A; Huri, H Z; Kamarul, T

    2016-01-14

    Underneath the intricacy of every cancer lies mysterious events that impel the tumour cell and its posterity into abnormal growth and tissue invasion. Oncogenic mutations disturb the regulatory circuits responsible for the governance of versatile cellular functions, permitting tumour cells to endure deregulated proliferation, resist to proapoptotic insults, invade and erode normal tissues and above all escape apoptosis. This disruption of apoptosis has been highly implicated in various malignancies and has been exploited as an anticancer strategy. Owing to the fact that apoptosis causes minimal inflammation and damage to the tissue, apoptotic cell death-based therapy has been the centre of attraction for the development of anticancer drugs. Increased understanding of the molecular pathways underlying apoptosis has enabled scientists to establish unique approaches targeting apoptosis pathways in cancer therapeutics. In this review, we reconnoitre the two major pathways (intrinsic and extrinsic) targeted cancer therapeutics, steering toward chief modulators of these pathways, such as B-cell lymphoma 2 protein family members (pro- and antiapoptotic), inhibitor of apoptosis proteins, and the foremost thespian of extrinsic pathway regulator, tumour necrosis factor-related apoptosis-inducing agent. Together, we also will have a look from clinical perspective to address the agents (drugs) and therapeutic strategies adopted to target these specific proteins/pathways that have entered clinical trials.

  12. Antcin B and its ester derivative from Antrodia camphorata induce apoptosis in hepatocellular carcinoma cells involves enhancing oxidative stress coincident with activation of intrinsic and extrinsic apoptotic pathway.

    PubMed

    Hsieh, Yun-Chih; Rao, Yerra Koteswara; Whang-Peng, Jacqueline; Huang, Chi-Ying F; Shyue, Song-Kun; Hsu, Shih-Lan; Tzeng, Yew-Min

    2011-10-26

    The triterpenoids methylantcinate B (MAB) and antcin B (AB), isolated from the medicinal mushroom Antrodia camphorata , have been identified as strong cytotoxic agents against various type of cancer cells; however, the mechanisms of MAB- and AB-induced cytotoxicity have not been adequately explored. This study investigated the roles of caspase cascades, reactive oxygen species (ROS), DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in MAB- and AB-induced apoptosis of hepatocellular carcinoma (HCC) HepG2 cells. Here, we showed that MAB and AB induced apoptosis in HepG2 cells, as characterized by increased DNA fragmentation, cleavage of PARP, sub-G1 population, chromatin condensation, loss of mitochondrial membrane potential, and release of cytochrome c. Increasing the levels of caspase-2, -3, -8, and -9 activities was involved in MAB- and AB-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that MAB and AB triggered the caspase-dependent apoptotic pathway. Additionally, the enhanced apoptotic effect correlates with high expression of Fas, Fas ligand, as well as Bax and decreased protein levels of Bcl-(XL) and Bcl-2, suggesting that both the extrinsic and intrinsic apoptosis pathways were involved in the apoptotic processes. Incubation of HepG2 cells with antioxidant enzymes superoxide dismutase and catalase and antioxidants N-acetylcysteine and ascorbic acid attenuated the ROS generation and apoptosis induced by MAB and AB, which indicate that ROS plays a pivotal role in cell death. NADPH oxidase activation was observed in MAB- and AB-stimulated HepG2 cells; however, inhibition of such activation by diphenylamine significantly blocked MAB- and AB-induced ROS production and increased cell viability. Taken together, our results provide the first evidence that triterpenoids MAB and AB induced a NADPH oxidase-provoked oxidative stress and extrinsic and intrinsic apoptosis as a critical mechanism of cause cell

  13. Discovery of Sulfonamidebenzamides as Selective Apoptotic CHOP Pathway Activators of the Unfolded Protein Response

    PubMed Central

    2014-01-01

    Cellular proteins that fail to fold properly result in inactive or disfunctional proteins that can have toxic functions. The unfolded protein response (UPR) is a two-tiered cellular mechanism initiated by eukaryotic cells that have accumulated misfolded proteins within the endoplasmic reticulum (ER). An adaptive pathway facilitates the clearance of the undesired proteins; however, if overwhelmed, cells trigger apoptosis by upregulating transcription factors such as C/EBP-homologous protein (CHOP). A high throughput screen was performed directed at identifying compounds that selectively upregulate the apoptotic CHOP pathway while avoiding adaptive signaling cascades, resulting in a sulfonamidebenzamide chemotype that was optimized. These efforts produced a potent and selective CHOP inducer (AC50 = 0.8 μM; XBP1 > 80 μM), which was efficacious in both mouse embryonic fibroblast cells and a human oral squamous cell cancer cell line, and demonstrated antiproliferative effects for multiple cancer cell lines in the NCI-60 panel. PMID:25530830

  14. Differential regulation of hepatic apoptotic pathways by dietary olive and sunflower oils in the aging rat.

    PubMed

    Bello, Rosario I; Gómez-Díaz, Consuelo; Burón, María I; Navas, Plácido; Villalba, José M

    2006-11-01

    In this work we have studied how dietary fat affects aging-related changes in a number of factors that regulate rat hepatic apoptosis. Animals were fed lifelong with two experimental diets containing either virgin olive oil or sunflower oil as dietary fat. Caspases of the intrinsic and extrinsic pathways of apoptosis, Bcl-2 and Bax polypeptide levels, and plasma membrane neutral sphingomyelinase activity were determined at 6, 12, and 24 months of age. Caspase-8/10 activity (a marker of the extrinsic pathway) was not affected by either aging or dietary fat, but activities of both caspase-9 (a marker of the intrinsic pathway) and caspase-3 (an executioner caspase) were significantly depressed in liver from animals fed on a sunflower oil-based diet. These decreases were not observed in animals fed with a diet based on virgin olive oil, which also resulted in significantly lower Bcl-2/Bax ratios. On the other hand, in comparison with sunflower, dietary olive oil decreased oxidative stress in liver from aged rats, resulting in lower levels of membrane hydroperoxides and higher coenzyme Q levels in plasma membrane. Plasma membrane Mg(2+)-dependent neutral sphingomyelinase was strongly activated in aged rats fed on the sunflower oil diet, but no aging-related increase was observed in animals fed on the olive oil diet. Our results support that dietary oil can alter significantly the susceptibility of hepatocytes to different apoptotic stimuli by altering both pro- and anti-apoptotic mediators, which reinforces the importance of the diet in aging studies. Because virgin olive oil may increase susceptibility of hepatocytes to apoptosis induced through the intrinsic pathway under conditions of decreased oxidative stress, our results may have important implications to understand the potential beneficial effects of that edible oil against liver carcinogenesis during aging.

  15. Mitochondria-specific accumulation of amyloid β induces mitochondrial dysfunction leading to apoptotic cell death.

    PubMed

    Cha, Moon-Yong; Han, Sun-Ho; Son, Sung Min; Hong, Hyun-Seok; Choi, Young-Ju; Byun, Jayoung; Mook-Jung, Inhee

    2012-01-01

    Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ(1-42) in HT22 cells using Aβ(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ(1-42) accumulation, which mimics the apoptosis process in exogenous Aβ(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ(1-42) accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads

  16. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    SciTech Connect

    Song, Shasha; Wang, Shuang; Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin; Zhu, Daling

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  17. Cystein cathepsin and Hsp90 activities determine the balance between apoptotic and necrotic cell death pathways in caspase-compromised U937 cells.

    PubMed

    Imre, Gergely; Dunai, Zsuzsanna; Petak, Istvan; Mihalik, Rudolf

    2007-10-01

    Caspase-inhibited cells induced to die may exhibit the traits of either apoptosis or necrosis or both, simultaneously. However, mechanisms regulating the commitment to these distinct forms of cell death are barely identified. We found that staurosporine induced both apoptotic and necrotic traits in U937 cells exposed to the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone. Morphology and flow cytometry revealed that individual cells exhibited either apoptotic or necrotic traits, but not the mixed phenotype. Inhibition of cathepsin activity by benzyloxycarbonyl-Phe-Ala-fluoromethylketone rendered caspase-compromised cells resistant to staurosporine-induced apoptosis, but switched the cell death form to necrosis. Inhibition of heat shock protein 90 kDa (Hsp90) chaperon activity by geldanamycin conferred resistance to necrosis in caspase-compromised cells but switched the cell death form to apoptosis. Combination of benzyloxycarbonyl-Phe-Ala-fluoromethylketone and geldanamycin halted the onset of both forms of cell death by saving mitochondrial trans-membrane potential and preventing acidic volume (lysosomes) loss. These effects of benzyloxycarbonyl-Phe-Ala-fluoromethylketone and/or geldanamycin on cell death were restricted to caspase-inhibited cells exposed to staurosporine but influenced neither only the staurosporine-provoked apoptosis nor hydrogen peroxide (H2O2)-generated necrosis. Our results demonstrate that the staurosporine-induced death pathway bifurcates in caspase-compromised cells and commitment to apoptotic or necrotic phenotypes depends on cathepsin protease or Hsp90 chaperon activities.

  18. Altered Transmission of HOX and Apoptotic SNPs Identify a Potential Common Pathway for Clubfoot

    PubMed Central

    Ester, Audrey R.; Weymouth, Katelyn S.; Burt, Amber; Wise, Carol; Scott, Allison; Gurnett, Christina A; Dobbs, Matthew B.; Blanton, Susan H.; Hecht, Jacqueline T.

    2009-01-01

    Clubfoot is a common birth defect that affects 135,000 newborns each year worldwide. It is characterized by equinus deformity of one or both feet and hypoplastic calf muscles. Despite numerous study approaches, the cause(s) remains poorly understood although a multifactorial etiology is generally accepted. We considered the HOXA and HOXD gene clusters and insulin-like growth factor binding protein 3 (IGFBP3) as candidate genes because of their important roles in limb and muscle morphogenesis. Twenty SNPs from the HOXA and HOXD gene clusters and 12 SNPs in IGFBP3 were genotyped in a sample composed of nonHispanic white and Hispanic multiplex and simplex families (discovery samples) and a second sample of nonHispanic white simplex trios (validation sample). Four SNPs (rs6668, rs2428431, rs3801776 and rs3779456) in the HOXA cluster demonstrated altered transmission in the discovery sample, but only rs3801776, located in the HOXA basal promoter region, showed altered transmission in both the discovery and validation samples (p=0.004 and p=0.028). Interestingly, HOXA9 is expressed in muscle during development. A SNP in IGFBP3, rs13223993, also showed altered transmission (p=0.003) in the discovery sample. Gene-gene interactions were identified between variants in HOXA, HOXD and IGFBP3 and with previously associated SNPs in mitochondrial-mediated apoptotic genes. The most significant interactions were found between CASP3 SNPS and variants in HOXA, HOXD and IGFBP3. These results suggest a biologic model for clubfoot in which perturbation of HOX and apoptotic genes together affect muscle and limb development, which may cause the downstream failure of limb rotation into a plantar grade position. PMID:19938081

  19. Upregulation of extrinsic apoptotic pathway in curcumin-mediated antiproliferative effect on human pancreatic carcinogenesis.

    PubMed

    Youns, Mahmoud; Fathy, Gihan Mahmoud

    2013-12-01

    Pancreatic cancer is one of the most lethal human cancers, with almost identical incidence and mortality rates. Curcumin, derived from the rhizome of Curcuma longa, has a long history of use as coloring agent and for a wide variety of disorders. Here, the antiproliferative activity of curcumin and its modulatory effect on gene expression of pancreatic cancer cell lines were investigated. The effect of curcumin on cellular proliferation and viability was monitored by sulphurhodamine B assay. Apoptotic effect was evaluated by flow cytometry and further confirmed by measuring amount of cytoplasmic histone-associated DNA fragments. Analysis of gene expression was performed with and without curcumin treatment using microarray expression profiling techniques. Array results were confirmed by real-time PCR. ingenuity pathway analysis (IPA) has been used to classify the list of differentially expressed genes and to indentify common biomarkergenes modulating the chemopreventive effect of curcumin. Results showed that curcumin induces growth arrest and apoptosis in pancreatic cancer cell lines. Its effect was more obvious on the highly COX-2 expressing cell line. Additionally, the expression of 366 and 356 cancer-related genes, involved in regulation of apoptosis, cell cycle, metastasis, was significantly altered after curcumin treatment in BxPC-3 and MiaPaCa-2 cells, respectively. Our results suggested that up-regulation of the extrinsic apoptotic pathway was among signaling pathways modulating the growth inhibitory effects of curcumin on pancreatic cancer cells. Curcumin effect was mediated through activation of TNFR, CASP 8, CASP3, BID, BAX, and down-regulation of NFκB, NDRG 1, and BCL2L10 genes.

  20. Anti-apoptotic role of the sonic hedgehog signaling pathway in the proliferation of ameloblastoma.

    PubMed

    Kanda, Shiori; Mitsuyasu, Takeshi; Nakao, Yu; Kawano, Shintaro; Goto, Yuichi; Matsubara, Ryota; Nakamura, Seiji

    2013-09-01

    Sonic hedgehog (SHH) signaling pathway is crucial to growth and patterning during organogenesis. Aberrant activation of the SHH signaling pathway can result in tumor formation. We examined the expression of SHH signaling molecules and investigated the involvement of the SHH pathway in the proliferation of ameloblastoma, the most common benign tumor of the jaws. We used immunohistochemistry on ameloblastoma specimens and immunocytochemistry and reverse transcription-PCR on the ameloblastoma cell line AM-1. We also used the inhibitors of SHH signaling, SHH neutralizing antibody and cyclopamine, to assess the effects of SHH on the proliferation of AM-1 cells. We detected expression of SHH, patched, GLI1, GLI2 and GLI3 in the ameloblastoma specimens and AM-1 cells. The proliferation of these cells was significantly inhibited in the presence of SHH neutralizing antibody or cyclopamine; this was confirmed by BrdU incorporation assays. Furthermore, in the presence of SHH neutralizing antibody, nuclear translocation of GLI1 and GLI2 was abolished, apoptosis was induced, BCL-2 expression decreased and BAX expression increased. Our results suggest that the SHH signaling pathway is constitutively active in ameloblastoma and plays an anti-apoptotic role in the proliferation of ameloblastoma cells through autocrine loop stimulation.

  1. Interaction of biologically active amines with mitochondria and their role in the mitochondrial-mediated pathway of apoptosis.

    PubMed

    Toninello, A; Salvi, M; Mondovì, B

    2004-09-01

    The natural polyamines spermine, spermidine and putrescine, polycationic molecules at physiological pH, interact with mitochondrial membranes at two specific binding sites exhibiting low affinity and high binding capacity. This binding represents the first step in the electrophoretic mechanism of polyamine transport into mitochondria. Spermine accumulated into the mitochondrial matrix is able to flow out by an electroneutral mechanism. This process promotes bi-directional transport of polyamines in and out of mitochondria, driven by electrical potential and pH gradient, respectively. Polyamines and biogenic amines are oxidized by cytosolic and mitochondrial amine oxidases with the production of hydrogen peroxide and aldehydes, both of which are involved in the induction and/or amplification of the mitochondrial permeability transition (MPT). This phenomenon, which provokes a bioenergetic collapse and redox catastrophe, is strongly inhibited by polyamines in isolated mitochondria. Monoamines also exhibit an inhibitory effect at higher concentrations, but at low concentrations behave as inducer agents. MPT is characterized by the opening of a channel, the transition pore, which permits non-specific bi-directional traffic of solutes across the inner membrane, leading to swelling of the organelle and release of cytochrome c and apoptosis-inducing factors. These proteins in turn activate the caspase-cascade, which triggers the apoptotic pathway. Depending on their cytosolic concentration, metabolic conditions and cell type, polyamines act as promoting, modulating or protective agents in mitochondrial-mediated apoptosis. While their protective effect could reflect inhibition of MPT and retention of cytochrome c, the promoting effect can be explained by the generation of reactive oxygen species that induce the opposite effect on MPT and cytochrome c release. Polyamines and other active amines can also participate in the regulation of apoptotic pathways by interacting with

  2. Ilexsaponin A attenuates ischemia-reperfusion-induced myocardial injury through anti-apoptotic pathway

    PubMed Central

    Wang, Fang; Qiang, Jiao; Liu, Pan; Zhang, Jun; Xu, Jin-Wen

    2017-01-01

    The protective effects of ilexsaponin A on ischemia-reperfusion-induced myocardial injury were investigated. Myocardial ischemia/reperfusion model was established in male Sprague–Dawley rats. Myocardial injury was evaluated by TTC staining and myocardial marker enzyme leakage. The in vitro protective potential of Ilexsaponin A was assessed on hypoxia/reoxygenation cellular model in neonatal rat cardiomyocytes. Cellular viability and apoptosis were evaluated by MTT and TUNEL assay. Caspase-3, cleaved caspase-3, bax, bcl-2, p-Akt and Akt protein expression levels were detected by western-blot. Ilexsaponin A treatment was able to attenuate the myocardial injury in ischemia/reperfusion model by reducing myocardial infarct size and lower the serum levels of LDH, AST and CK-MB. The in vitro study also showed that ilexsaponin A treatment could increase cellular viability and inhibit apoptosis in hypoxia/reoxygenation cardiomyocytes. Proapoptotic proteins including caspase-3, cleaved caspase-3 and bax were significantly reduced and anti-apoptotic protein bcl-2 was significantly increased by ilexsaponin A treatment in hypoxia/reoxygenation cardiomyocytes. Moreover, Ilexsaponin A treatment was able to increase the expression levels of p-Akt in hypoxia/reoxygenation cellular model and myocardial ischemia/reperfusion animal model. Coupled results from both in vivo and in vitro experiments indicate that Ilexsaponin A attenuates ischemia-reperfusion-induced myocardial injury through anti-apoptotic pathway. PMID:28182689

  3. The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes.

    PubMed Central

    Chuang, T H; Hahn, K M; Lee, J D; Danley, D E; Bokoch, G M

    1997-01-01

    Apoptosis plays an important role in regulating development and homeostasis of the immune system, yet the elements of the signaling pathways that control cell death have not been well defined. When expressed in Jurkat T cells, an activated form of the small GTPase Cdc42 induces cell death exhibiting the characteristics of apoptosis. The death response induced by Cdc42 is mediated by activation of a protein kinase cascade leading to stimulation of c-Jun amino terminal kinase (JNK). Apoptosis initiated by Cdc42 is inhibited by dominant negative components of the JNK cascade and by reagents that block activity of the ICE protease (caspase) family, suggesting that stimulation of the JNK kinase cascade can lead to caspase activation. The sequence of morphological events observed typically in apoptotic cells is modified in the presence of activated Cdc42, suggesting that this GTPase may account for some aspects of cytoskeletal regulation during the apoptotic program. These data suggest a means through which the biochemical and morphological events occurring during apoptosis may be coordinately regulated. Images PMID:9307966

  4. Gold nanoparticles enhance TRAIL sensitivity through Drp1-mediated apoptotic and autophagic mitochondrial fission in NSCLC cells

    PubMed Central

    Ke, Sunkui; Zhou, Tong; Yang, Peiyan; Wang, Yange; Zhang, Peng; Chen, Keman; Ren, Lei; Ye, Shefang

    2017-01-01

    Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its agonistic receptors have been identified as highly promising antitumor agents preferentially eliminating cancer cells with minimal damage, the emergence of TRAIL resistance in most cancers may contribute to therapeutic failure. Thus, there is an urgent need for new approaches to overcome TRAIL resistance. Gold nanoparticles (AuNPs) are one of the most promising nanomaterials that show immense antitumor potential via targeting various cellular and molecular processes; however, the effects of AuNPs on TRAIL sensitivity in cancer cells remain unclear. In this study, we found that AuNPs combined with TRAIL exhibited a greater potency in promoting apoptosis in non-small-cell lung cancer (NSCLC) cells compared with TRAIL alone, suggesting that AuNPs sensitize cancer cells to TRAIL. Further experiments demonstrated that the combination of TRAIL and AuNPs was more effective in causing excessive mitochondrial fragmentation in cancer cells accompanied by a dramatic increase in mitochondrial recruitment of dynamin-related protein 1 (Drp1), mitochondrial dysfunctions, and enhancement of autophagy induction. Small interfering RNA (siRNA) silencing of Drp1 or inhibition of autophagy could effectively alleviate apoptosis in cells exposed to TRAIL combined with AuNPs. In vivo studies revealed that AuNPs augmented TRAIL sensitivity in tumor-bearing mice. Our data indicated that AuNPs potentiate apoptotic response to TRAIL in NSCLC cells through Drp1-dependent mitochondrial fission, and TRAIL combined with AuNPs can be a potential chemotherapeutic strategy for the treatment of NSCLC.

  5. Apoptotic transition of senescent cells accompanied with mitochondrial hyper-function

    PubMed Central

    Wang, Danli; Liu, Yang; Zhang, Rui; Zhang, Fen; Sui, Weihao; Chen, Li; Zheng, Ran; Chen, Xiaowen; Wen, Feiqiu; Ouyang, Hong-Wei; Ji, Junfeng

    2016-01-01

    Defined as stable cell-cycle arrest, cellular senescence plays an important role in diverse biological processes including tumorigenesis, organismal aging, and embryonic development. Although increasing evidence has documented the metabolic changes in senescent cells, mitochondrial function and its potential contribution to the fate of senescent cells remain largely unknown. Here, using two in vitro models of cellular senescence induced by doxorubicin treatment and prolonged passaging of neonatal human foreskin fibroblasts, we report that senescent cells exhibited high ROS level and augmented glucose metabolic rate concomitant with both morphological and quantitative changes of mitochondria. Furthermore, mitochondrial membrane potential depolarized at late stage of senescent cells which eventually led to apoptosis. Our study reveals that mitochondrial hyper-function contributes to the implementation of cellular senescence and we propose a model in which the mitochondrion acts as the key player in promoting fate-determination in senescent cells. PMID:27056883

  6. A new pathway for mitochondrial quality control: mitochondrial-derived vesicles.

    PubMed

    Sugiura, Ayumu; McLelland, Gian-Luca; Fon, Edward A; McBride, Heidi M

    2014-10-01

    The last decade has been marked by tremendous progress in our understanding of the cell biology of mitochondria, with the identification of molecules and mechanisms that regulate their fusion, fission, motility, and the architectural transitions within the inner membrane. More importantly, the manipulation of these machineries in tissues has provided links between mitochondrial dynamics and physiology. Indeed, just as the proteins required for fusion and fission were identified, they were quickly linked to both rare and common human diseases. This highlighted the critical importance of this emerging field to medicine, with new hopes of finding drugable targets for numerous pathologies, from neurodegenerative diseases to inflammation and cancer. In the midst of these exciting new discoveries, an unexpected new aspect of mitochondrial cell biology has been uncovered; the generation of small vesicular carriers that transport mitochondrial proteins and lipids to other intracellular organelles. These mitochondrial-derived vesicles (MDVs) were first found to transport a mitochondrial outer membrane protein MAPL to a subpopulation of peroxisomes. However, other MDVs did not target peroxisomes and instead fused with the late endosome, or multivesicular body. The Parkinson's disease-associated proteins Vps35, Parkin, and PINK1 are involved in the biogenesis of a subset of these MDVs, linking this novel trafficking pathway to human disease. In this review, we outline what has been learned about the mechanisms and functional importance of MDV transport and speculate on the greater impact of these pathways in cellular physiology.

  7. A new pathway for mitochondrial quality control: mitochondrial-derived vesicles

    PubMed Central

    Sugiura, Ayumu; McLelland, Gian-Luca; Fon, Edward A; McBride, Heidi M

    2014-01-01

    The last decade has been marked by tremendous progress in our understanding of the cell biology of mitochondria, with the identification of molecules and mechanisms that regulate their fusion, fission, motility, and the architectural transitions within the inner membrane. More importantly, the manipulation of these machineries in tissues has provided links between mitochondrial dynamics and physiology. Indeed, just as the proteins required for fusion and fission were identified, they were quickly linked to both rare and common human diseases. This highlighted the critical importance of this emerging field to medicine, with new hopes of finding drugable targets for numerous pathologies, from neurodegenerative diseases to inflammation and cancer. In the midst of these exciting new discoveries, an unexpected new aspect of mitochondrial cell biology has been uncovered; the generation of small vesicular carriers that transport mitochondrial proteins and lipids to other intracellular organelles. These mitochondrial-derived vesicles (MDVs) were first found to transport a mitochondrial outer membrane protein MAPL to a subpopulation of peroxisomes. However, other MDVs did not target peroxisomes and instead fused with the late endosome, or multivesicular body. The Parkinson's disease-associated proteins Vps35, Parkin, and PINK1 are involved in the biogenesis of a subset of these MDVs, linking this novel trafficking pathway to human disease. In this review, we outline what has been learned about the mechanisms and functional importance of MDV transport and speculate on the greater impact of these pathways in cellular physiology. PMID:25107473

  8. Tris (1,3-dichloro-2-propyl) phosphate-induced apoptotic signaling pathways in SH-SY5Y neuroblastoma cells.

    PubMed

    Li, Ruiwen; Zhou, Peijiang; Guo, Yongyong; Lee, Jae-Seong; Zhou, Bingsheng

    2017-01-01

    Tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP, also known as TDCPP), an extensively used flame retardant, is frequently detected in the environment and biota. Recent studies have shown that TDCIPP has neurotoxic effects. In this study, we determined the mechanisms of TDCIPP-induced neurotoxicity in human neuroblastoma (SH-SY5Y) cells. By using morphological examination, flow cytometry, and mitochondrial membrane potential (ΔYm) measurement, we confirmed that exposure to TDCIPP caused apoptosis accompanied by the activation of apoptosis-related genes (e.g. Bax and Bcl-2) and caspase 3 protein in SH-SY5Y cells. Increased reactive oxygen species (ROS) formation and intracellular calcium ions ([Ca(2+)]i) were also observed in TDCIPP-treated SH-SY5Y cells. Exposure to TDCIPP led to the activation of protein markers of endoplasmic reticulum (ER) stress, including eukaryotic translation initiation factor 2a subunit (p-EIF2a), activation transcription factor (ATF4), glucose-regulated protein (GRP78), and the proapoptotic factor C/EBP homologous protein (CHOP). To determine the role of the ER in apoptosis, phenyl butyric acid (PBA), an ER stress inhibitor, was applied. Treatment with PBA effectively attenuated TDCIPP-induced ER stress and protected against apoptotic death in SH-SY5Y cells by inhibition of Bax expression and promotion of Bcl-2 expression. Furthermore, we found that pretreatment of the cells with the ROS scavenger N-acetyl cysteine (NAC) inhibited the ER stress response and prevented apoptosis. The combination of PBA and NAC pretreatment could further prevent TDCIPP induced ER-stress and apoptotic death compared with PBA or NAC pretreatment alone. Thus, in the present study, we demonstrated that TDCIPP induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathways in SH-SY5Y cells.

  9. Phloroglucinol induces apoptosis via apoptotic signaling pathways in HT-29 colon cancer cells

    PubMed Central

    KANG, MI-HYE; KIM, IN-HYE; NAM, TAEK-JEO NG

    2014-01-01

    Phloroglucinol is a polyphenolic compound that is used to treat and prevent several human diseases, as it exerts beneficial biological activities, including anti-oxidant, anti-inflammatory and anticancer properties. The aim of the present study was to investigate the effects of phloroglucinol on apoptotic signaling pathways in HT-29 colon cancer cells. The results indicated that phloroglucinol suppressed cell viability and induced apoptosis in HT-29 cells in a concentration-dependent manner. Phloroglucinol treatment of HT-29 cells resulted in characteristic apoptosis-related changes: altered Bcl-2 family proteins, cytochrome c release, and activation of caspase-3 and caspase-8. This study also showed that proteins involved in apoptosis were stimulated by treatment with phloroglucinol. These findings demonstrated that phloroglucinol exerts anticancer activity in HT-29 colon cancer cells through induction of apoptosis. PMID:25070748

  10. Molecular Pathways: Mitochondrial Reprogramming in Tumor Progression and Therapy

    PubMed Central

    Caino, M. Cecilia; Altieri, Dario C.

    2015-01-01

    Small molecule inhibitors of the phosphatidylinositol 3-kinase (PI3K), Akt and mTOR pathway currently in the clinic produce a paradoxical reactivation of the pathway they are intended to suppress. Furthermore, fresh experimental evidence with PI3K antagonists in melanoma, glioblastoma and prostate cancer shows that mitochondrial metabolism drives an elaborate process of tumor adaptation culminating with drug resistance and metastatic competency. This is centered on reprogramming of mitochondrial functions to promote improved cell survival and to fuel the machinery of cell motility and invasion. Key players in these responses are molecular chaperones of the Heat Shock Protein 90 (Hsp90) family compartmentalized in mitochondria, which suppress apoptosis via phosphorylation of the pore component, Cyclophilin D, and enable the subcellular repositioning of active mitochondria to membrane protrusions implicated in cell motility. An inhibitor of mitochondrial Hsp90s in preclinical development (Gamitrinib) prevents adaptive mitochondrial reprogramming and shows potent anti-tumor activity in vitro and in vivo. Other therapeutic strategies to target mitochondria for cancer therapy include small molecule inhibitors of mutant isocitrate dehydrogenase (IDH) IDH1 (AG-120) and IDH2 (AG-221) which opened new therapeutic prospects for high-risk AML patients. A second approach of mitochondrial therapeutics focuses on agents that elevate toxic ROS levels from a leaky electron transport chain, nevertheless the clinical experience with these compounds, including a quinone derivative, ARQ 501, and a copper chelator, elesclomol (STA-4783) is limited. In light of these evidences, we discuss how best to target a resurgence of mitochondrial bioenergetics for cancer therapy. PMID:26660517

  11. The extrinsic apoptotic signaling pathway in gastric adenocarcinomas assessed by tissue microarray.

    PubMed

    Gomes, Thiago S; Oshima, Celina T F; Segreto, Helena R C; Barrazueta, Luis M; Costa, Henrique O; Lima, Flavio O; Forones, Nora M; Ribeiro, Daniel A

    2011-10-15

    The purpose of this investigation was to analyze the immunoexpression of FasL, Fas, FADD, cleaved caspase 8, and cleaved caspase 3 in gastric cancer. Formalin-fixed and paraffin-embedded gastric adenocarcinoma tissues from 87 patients, including adjacent normal tissues, were included on tissue microarray by immunohistochemistry. The tumor and the adjacent normal tissues were positive for FasL in 66.7% and 90.6%, for Fas in 52.8% and 52.4%, for FADD in 67.4% and 82.3%, for cleaved caspase 8 in 27.9% and 37.7%, and for cleaved caspase 3 in 33.7% and 8.3%, respectively. FasL and the FADD from tumor were statistically different in relation to the histological type. Cleaved caspase 8 was statistically different in relation to clinical stage (p=0.031). The FADD from normal tissue was statistically different in relation to age (p=0.039), sex (p=0.055), clinical stage (p=0.019), and Fas was different in relation to tumor size (p=0.012). In the tumor, we observed a correlation between FasL and Fas, FasL and FADD, and FasL and cleaved caspase 3. In the adjacent normal tissue, a correlation was observed between FasL and Fas, FasL and FADD. There was no association of another marker with sex, age, clinical stage, and survival. Our results suggest that these proteins mediate the early extrinsic apoptotic pathway in gastric cancer and adjacent normal mucosa. FasL protein binds to Fas protein and subsequently binds to death receptor FADD signaling activation of the extrinsic apoptotic pathway. In this phase, there was inhibition of caspase 8 and, consequently, decreased apoptosis.

  12. Neuronal apoptotic signaling pathways probed and intervened by synthetically and modularly modified (SMM) chemokines.

    PubMed

    Choi, Won-Tak; Kaul, Marcus; Kumar, Santosh; Wang, Jun; Kumar, I M Krishna; Dong, Chang-Zhi; An, Jing; Lipton, Stuart A; Huang, Ziwei

    2007-03-09

    As the main coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, CXCR4 and CCR5 play important roles in HIV-associated dementia (HAD). HIV-1 glycoprotein gp120 contributes to HAD by causing neuronal damage and death, either directly by triggering apoptotic pathways or indirectly by stimulating glial cells to release neurotoxins. Here, to understand the mechanism of CXCR4 or CCR5 signaling in neuronal apoptosis associated with HAD, we have applied synthetically and modularly modified (SMM)-chemokine analogs derived from natural stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II as chemical probes of the mechanism(s) whereby these SMM-chemokines prevent or promote neuronal apoptosis. We show that inherently neurotoxic natural ligands of CXCR4, such as stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II, can be modified to protect neurons from apoptosis induced by CXCR4-preferring gp120(IIIB), and that the inhibition of CCR5 by antagonist SMM-chemokines, unlike neuroprotective CCR5 natural ligands, leads to neurotoxicity by activating a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Furthermore, we discover distinct signaling pathways activated by different chemokine ligands that are either natural agonists or synthetic antagonists, thus demonstrating a chemical biology strategy of using chemically engineered inhibitors of chemokine receptors to study the signaling mechanism of neuronal apoptosis and survival.

  13. Proteasomal Dysfunction Induced By Diclofenac Engenders Apoptosis Through Mitochondrial Pathway.

    PubMed

    Amanullah, Ayeman; Upadhyay, Arun; Chhangani, Deepak; Joshi, Vibhuti; Mishra, Ribhav; Yamanaka, Koji; Mishra, Amit

    2017-05-01

    Diclofenac is the most commonly used phenylacetic acid derivative non-steroidal anti-inflammatory drug (NSAID) that demonstrates significant analgesic, antipyretic, and anti-inflammatory effects. Several epidemiological studies have demonstrated anti-proliferative activity of NSAIDs and examined their apoptotic induction effects in different cancer cell lines. However, the precise molecular mechanisms by which these pharmacological agents induce apoptosis and exert anti-carcinogenic properties are not well known. Here, we have observed that diclofenac treatment induces proteasome malfunction and promotes accumulation of different critical proteasome substrates, including few pro-apoptotic proteins in cells. Exposure of diclofenac consequently elevates aggregation of various ubiquitylated misfolded proteins. Finally, we have shown that diclofenac treatment promotes apoptosis in cells, which could be because of mitochondrial membrane depolarization and cytochrome c release into cytosol. This study suggests possible beneficial insights of NSAIDs-induced apoptosis that may improve our existing knowledge in anti-proliferative interspecific strategies development. J. Cell. Biochem. 118: 1014-1027, 2017. © 2016 Wiley Periodicals, Inc.

  14. Sanguisorba officinalis L synergistically enhanced 5-fluorouracil cytotoxicity in colorectal cancer cells by promoting a reactive oxygen species-mediated, mitochondria-caspase-dependent apoptotic pathway

    PubMed Central

    Liu, Meng-ping; Liao, Min; Dai, Cong; Chen, Jie-feng; Yang, Chun-juan; Liu, Ming; Chen, Zuan-guang; Yao, Mei-cun

    2016-01-01

    Sanguisorba officinalis L. radix is a widely used herb called DiYu (DY) in China and has an extensive range of bioactivities, including anti-cancer, anti-inflammatory, and anti-oxidative activities. However, there is little evidence to support its anti-cancer effects against colorectal cancer (CRC). The first-line chemotherapeutic agent 5-fluorouracil (5-FU) is used to treat CRC, but its efficiency is hampered by acquired drug resistance. This study found that a water extract of DY exerted anti-proliferative effects against two CRC cell lines (HCT-116 and RKO), and it sensitized CRC cells to 5-FU therapy by activating a reactive oxygen species (ROS)-mediated, mitochondria-caspase-dependent apoptotic pathway. Co-treatment of DY and 5-FU significantly elevated ROS levels, up-regulated Bax/Bcl-2 ratio and triggered mitochondrial dysfunction, followed by a release of cytochrome c and up-regulation of proteins such as cleaved-caspase-9/3 and cleaved-PARP. Additionally, the induction of autophagy may be involved in mediating synergism of DY in HCT-116 cells. Gallic acid (GA), catechinic acid (CA) and ellagic acid (EA) were identified as the potential chief constituents responsible for the synergistic effects of DY. In conclusion, co-treatment of DY, specifically GA, CA and EA, with 5-FU may be a potential alternative therapeutic strategy for CRC by enhancing an intrinsic apoptotic pathway. PMID:27671231

  15. Erastin Disrupts Mitochondrial Permeability Transition Pore (mPTP) and Induces Apoptotic Death of Colorectal Cancer Cells

    PubMed Central

    Huo, Haizhong; Zhou, Zhiyuan; Qin, Jian; Liu, Wenyong; Wang, Bing; Gu, Yan

    2016-01-01

    We here evaluated the potential anti-colorectal cancer activity by erastin, a voltage-dependent anion channel (VDAC)-binding compound. Our in vitro studies showed that erastin exerted potent cytotoxic effects against multiple human colorectal cancer cell lines, possibly via inducing oxidative stress and caspase-9 dependent cell apoptosis. Further, mitochondrial permeability transition pore (mPTP) opening was observed in erastin-treated cancer cells, which was evidenced by VDAC-1 and cyclophilin-D (Cyp-D) association, mitochondrial depolarization, and cytochrome C release. Caspase inhibitors, the ROS scavenger MnTBAP, and mPTP blockers (sanglifehrin A, cyclosporin A and bongkrekic acid), as well as shRNA-mediated knockdown of VDAC-1, all significantly attenuated erastin-induced cytotoxicity and apoptosis in colorectal cancer cells. On the other hand, over-expression of VDAC-1 augmented erastin-induced ROS production, mPTP opening, and colorectal cancer cell apoptosis. In vivo studies showed that intraperitoneal injection of erastin at well-tolerated doses dramatically inhibited HT-29 xenograft growth in severe combined immunodeficient (SCID) mice. Together, these results demonstrate that erastin is cytotoxic and pro-apoptotic to colorectal cancer cells. Erastin may be further investigated as a novel anti-colorectal cancer agent. PMID:27171435

  16. Antimicrobial Peptide-induced Apoptotic Death of Leishmania Results from Calcium-de pend ent, Caspase-independent Mitochondrial Toxicity*

    PubMed Central

    Kulkarni, Manjusha M.; Robert McMaster, W.; Kamysz, Wojciech; McGwire, Bradford S.

    2009-01-01

    α- and θ-defensin-, magainin-, and cathelicidin-type antimicrobial peptides (AMPs) can kill the pathogenic protozoan Leishmania. Comparative studies of a panel of AMPs have defined two distinct groups: those that induce nonapoptotic (Class I) and apoptotic (Class II) parasite killing based on their differential ability to induce phosphatidyl serine exposure, loss of mitochondrial membrane potential and decreased ATP production, induction of caspase-3/7 and -12 activity, and DNA degradation. Class II AMPs cause rapid influx of the vital stain SYTOX and an increase in intracellular Ca2+, whereas Class I AMPs cause a slow accumulation of SYTOX and do not affect intracellular Ca2+ levels. Inhibitors of cysteine or caspase proteases diminished fast influx of SYTOX through the surface membrane and DNA degradation but do not ablate the annexin V staining or the induction of apoptosis by Class II AMPs. This suggests that the changes in surface permeability in AMP-mediated apoptosis are related to the downstream events of intracellular cysteine/caspase activation or the loss of ATP. The activation of caspase-12-like activity was Ca2+-dependent, and inhibitors of voltage-gated and nonspecific Ca2+ channels diminished this activity. Flufenamic acid, a nonspecific Ca2+ inhibitor, completely ablated AMP-induced mitochondrial dysfunction and cell death, indicating the importance of dysregulation of Ca2+ in antimicrobial peptide-induced apoptosis. PMID:19357081

  17. The pivotal role of the mitochondrial amidoxime reducing component 2 in protecting human cells against apoptotic effects of the base analog N6-hydroxylaminopurine.

    PubMed

    Plitzko, Birte; Havemeyer, Antje; Kunze, Thomas; Clement, Bernd

    2015-04-17

    N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.

  18. In vitro analysis of the role of the mitochondrial apoptosis pathway in CSBE therapy against human gastric cancer

    PubMed Central

    JI, YU-BIN; YU, LEI

    2015-01-01

    The caper plant (Capparis spinosa L.) was a common Uyghur folk medicine, and is a member of the Capparidaceae family. In a previous study, the n-butanol extract of C. spinosa L. (CSBE) was demonstrated to exert anti-tumor activity; however, the underlying mechanism is currently not understood. The present study aimed to elucidate the mechanism underlying the CSBE-induced mitochondrial apoptotic pathway, in order to investigate the anti-tumor effects of this plant extract. CSBE-induced apoptosis of the SGC-7901 human gastric cancer cell line was observed, and alterations in the expression levels and localization of initiators, markers, and executors of the mitochondrial apoptosis pathway were analyzed. Following treatment of SGC-7901 cells with CBSE, proliferation was inhibited and apoptosis was induced; and these effects were associated with mitochondrial membrane potential disruption, cytochrome c release into the cytoplasm, and caspase-9 and caspase-3 activation. CSBE may have induced SGC-7901 cell apoptosis by upregulating the expression of B-cell lymphoma-2 (BCL-2)-associated X protein, and downregulating the expression of BCL-2. The results of the present study suggested that CSBE may induce SGC-7901 cell apoptosis via activation of the mitochondrial apoptosis pathway. PMID:26668648

  19. Rasagiline and selegiline suppress calcium efflux from mitochondria by PK11195-induced opening of mitochondrial permeability transition pore: a novel anti-apoptotic function for neuroprotection.

    PubMed

    Wu, Yuqiu; Kazumura, Kimiko; Maruyama, Wakako; Osawa, Toshihiko; Naoi, Makoto

    2015-10-01

    Rasagiline and selegiline, inhibitors of type B monoamine oxidase (MAO-B), protect neurons from cell death in cellular and animal models. Suppression of mitochondrial membrane permeabilization and subsequent activation of apoptosis cascade, and induction of anti-apoptotic, pro-survival genes are proposed to contribute the anti-apoptotic function. Rasagiline suppresses neurotoxin- and oxidative stress-induced membrane permeabilization in isolated mitochondria, but the mechanism has been not fully clarified. In this paper, regulation of the mitochondrial permeability transition pore by rasagiline and selegiline was examined in apoptosis induced by PK11195, a ligand of the outer membrane translocator protein 18 kDa (TSPO) in SH-SY5Y cells. The pore opening was quantitatively measured using a simultaneous monitoring system for calcium (Ca(2+)) and superoxide (O2(-)) (Ishibashi et al. in Biochem Biophys Res Commun 344:571-580, 2006). The association of the pore opening with Ca(2+) efflux and ROS increase was proved by the inhibition of Bcl-2 overexpression and cyclosporine A treatment. Potency to release Ca(2+) was correlated with the cytotoxicity of TSPO antagonists, PK11195, FGIN-1-27 and protoporphyrin IX, whereas a TSPO agonist, 4-chloro-diazepamine, did not significantly increase Ca(2+) or cause cell death. Rasagiline and selegiline inhibited mitochondrial Ca(2+) efflux through the mitochondrial permeability transition pore dose dependently. Ca(2+) efflux was confirmed as the initial signal in mitochondrial apoptotic cascade, and the suppression of Ca(2+) efflux may account for the neuroprotective function of rasagiline and selegiline. The quantitative measurement of Ca(2+) efflux can be applied to determine anti-apoptotic activity of neuroprotective compounds. The role of mitochondrial Ca(2+) release in neuronal death and also in neuroprotection by MAO-B inhibitors is discussed.

  20. 2-Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway.

    PubMed

    Schweikl, Helmut; Petzel, Christine; Bolay, Carola; Hiller, Karl-Anton; Buchalla, Wolfgang; Krifka, Stephanie

    2014-03-01

    Resin monomers of dental composites like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation of dental pulp-derived cells, or induce cell death via apoptosis. The induction of apoptosis is related to the availability of the antioxidant glutathione, although a detailed understanding of the signaling pathways is still unknown. The present study provides insight into the causal relationship between oxidative stress, oxidative DNA damage, and the specific signaling pathway leading to HEMA-induced apoptosis in RAW264.7 mouse macrophages. The differential expression of the antioxidative enzymes superoxide dismutase, glutathione peroxidase, and catalase in HEMA-exposed cells indicated oxidative stress, which was associated with the cleavage of pro-caspase 3 as a critical apoptosis executioner. A 2-fold increase in the amount of mitochondrial superoxide anions after a 24 h exposure to HEMA (6-8 mM) was paralleled by a considerable decrease in the mitochondrial membrane potential (MMP). Additionally, expression of proteins critical for the signaling of apoptosis through the intrinsic mitochondrial pathway was detected. Transcription-dependent and transcription-independent mechanisms of p53-regulated apoptosis were activated, and p53 was translocated from the cytosol to mitochondria. HEMA-induced transcriptional activity of p53 was indicated by increased levels of PUMA localized to mitochondria as a potent inducer of apoptosis. The expression of Bcl-xL and Bax suggested that cells responded to stress caused by HEMA via the activation of a complicated and antagonistic machinery of pro- and anti-apoptotic Bcl-2 family members. A HEMA-induced and oxidative stress-sensitive delay of the cell cycle, indicating a DNA damage response, occurred independent of the influence of KU55399, a potent inhibitor of ATM (ataxia-telangiectasia mutated) activity. However, ATM, a protein kinase which

  1. Dysregulation of the intrinsic apoptotic pathway mediates megakaryocytic hyperplasia in myeloproliferative neoplasms

    PubMed Central

    Malherbe, Jacques A J; Fuller, Kathryn A; Mirzai, Bob; Kavanagh, Simon; So, Chi-Chiu; Ip, Ho-Wan; Guo, Belinda B; Forsyth, Cecily; Howman, Rebecca; Erber, Wendy N

    2016-01-01

    Aims Megakaryocyte expansion in myeloproliferative neoplasms (MPNs) is due to uncontrolled proliferation accompanied by dysregulation of proapoptotic and antiapoptotic mechanisms. Here we have investigated the intrinsic and extrinsic apoptotic pathways of megakaryocytes in human MPNs to further define the mechanisms involved. Methods The megakaryocytic expression of proapoptotic caspase-8, caspase-9, Diablo, p53 and antiapoptotic survivin proteins was investigated in bone marrow specimens of the MPNs (n=145) and controls (n=15) using immunohistochemistry. The megakaryocyte percentage positivity was assessed by light microscopy and correlated with the MPN entity, JAK2V617F/CALR mutation status and platelet count. Results The proportion of megakaryocytes in the MPNs expressing caspase-8, caspase-9, Diablo, survivin and p53 was significantly greater than controls. A greater proportion of myeloproliferative megakaryocytes expressed survivin relative to its reciprocal inhibitor, Diablo. Differences were seen between myelofibrosis, polycythaemia vera and essential thrombocythaemia for caspase-9 and p53. CALR-mutated cases had greater megakaryocyte p53 positivity compared to those with the JAK2V617F mutation. Proapoptotic caspase-9 expression showed a positive correlation with platelet count, which was most marked in myelofibrosis and CALR-mutated cases. Conclusions Disruptions targeting the intrinsic apoptotic cascade promote megakaryocyte hyperplasia and thrombocytosis in the MPNs. There is progressive dysfunction of apoptosis as evidenced by the marked reduction in proapoptotic caspase-9 and accumulation of p53 in myelofibrosis. The dysfunction of caspase-9, which is necessary for proplatelet formation, may be the mechanism for the excess thrombocytosis associated with CALR mutations. Survivin seems to be the key protein mediating the megakaryocyte survival signature in the MPNs and is a potential therapeutic target. PMID:27060176

  2. Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage: Role of NF-{kappa}B, p38 and JNK MAPK pathway

    SciTech Connect

    Ghosh, Jyotirmoy; Das, Joydeep; Manna, Prasenjit

    2009-10-01

    Cardiac dysfunction is a major cause of morbidity and mortality worldwide due to its complex pathogenesis. However, little is known about the mechanism of arsenic-induced cardiac abnormalities and the use of antioxidants as the possible protective agents in this pathophysiology. Conditionally essential amino acid, taurine, accounts for 25% to 50% of the amino acid pool in myocardium and possesses antioxidant properties. The present study has, therefore, been carried out to investigate the underlying mechanism of the beneficial role of taurine in arsenic-induced cardiac oxidative damage and cell death. Arsenic reduced cardiomyocyte viability, increased reactive oxygen species (ROS) production and intracellular calcium overload, and induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-{kappa}B (p65) phosphorylation. Pre-exposure of myocytes to an IKK inhibitor (PS-1145) prevented As-induced caspase-3 and PARP cleavage. Arsenic also markedly increased the activity of p38 and JNK MAPKs, but not ERK to that extent. Pre-treatment with SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated NF-{kappa}B and IKK phosphorylation indicating that p38 and JNK MAPKs are mainly involved in arsenic-induced NF-{kappa}B activation. Taurine treatment suppressed these apoptotic actions, suggesting that its protective role in arsenic-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways. Similarly, arsenic intoxication altered a number of biomarkers related to cardiac oxidative stress and other apoptotic indices in vivo and taurine supplementation could reduce it. Results suggest that taurine prevented arsenic-induced myocardial pathophysiology, attenuated NF-{kappa}B activation via IKK, p38 and JNK MAPK signaling pathways and could possibly provide a protection

  3. Heat shock induces apoptosis through reactive oxygen species involving mitochondrial and death receptor pathways in corneal cells.

    PubMed

    Hsu, Ya-Ling; Yu, Hsin-Su; Lin, Hsien-Chung; Wu, Kwou-Yeung; Yang, Rei-Cheng; Kuo, Po-Lin

    2011-10-01

    Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Here, we are the first to investigate the biological effect of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. Our results indicate that heat shock exhibits effective cell proliferation inhibition by inducing apoptosis. Heat shock triggers the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activity. In addition, heat shock triggered the death receptor apoptotic pathway indicated by a change in Fas ligand expression, resulting in caspase-8 activity. Furthermore, we also found that generation of reactive oxygen species (ROS) is a critical mediator in heat shock-induced apoptosis. In addition, the antioxidant vitamin C significantly decreased heat shock-mediated apoptosis. Taken together, these findings suggest a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells.

  4. Sine Oculis Homeobox Homolog 1 Regulates Mitochondrial Apoptosis Pathway Via Caspase-7 In Gastric Cancer Cells

    PubMed Central

    Du, Peizhun; Zhao, Jing; Wang, Jing; Liu, Yongchao; Ren, Hong; Patel, Rajan; Hu, Cheng'en; Zhang, Wenhong; Huang, Guangjian

    2017-01-01

    Sine oculis homeobox homolog 1 (Six1) is crucial in normal organ development. Recently, Six1 is reported to display aberrant expression in various cancers and plays important roles in cancer development. However, the regulatory mechanism of Six1 in gastric cancer is largely unknown. In the current study, we found that Six1 was increased in gastric cancer tissues, and its upregulation significantly associated with lymph node metastasis (p=0.042) and poor differentiation (p=0.039). Next, we took advantage of public available microarray data to assess Six1 prognostic value with online K-M Plotter software in gastric cancer, which demonstrated that patients with higher Six1 expression had shorter survival time (p=0.02). To explore the underlying mechanism of Six1, we silenced its upregulation in gastric cells to detect cellular functions. Our results indicated that knock-down Six1 could decrease colony formation number and rendered cells sensitive to 5- Fluorouracil drug treatment. The flow cytometry analyses showed that Six1 silence could promote apoptosis but had little effect on cell cycle transition. Along this clue, we tested mitochondrial membrane potential with JC-1 assay, which suggested that Six1 inhibition could trigger mitochondrial apoptosis. Our subsequent results revealed that Six1 knock-down could reduce the level of anti-apoptotic protein Bcl-2, and caspase-7 but not caspase-3 was involved to execute the mitochondrial apoptosis pathway. Taken together, we find Six1 has oncogenic role in gastric cancer development, and silenced Six1 expression can promote mitochondrial apoptosis by repressing Bcl-2 and activating executor caspase-7. These findings suggest that Six1 may become a valuable prognostic and therapeutic target in gastric cancer. PMID:28367243

  5. Apoptotic mitochondrial dysfunction induced by benzo(a)pyrene in liver epithelial cells: role of p53 and pHi changes.

    PubMed

    Huc, Laurence; Gilot, David; Gardyn, Claire; Rissel, Mary; Dimanche-Boitrel, Marie-Thérèse; Guillouzo, André; Fardel, Olivier; Lagadic-Gossmann, Dominique

    2003-12-01

    How pH(i) changes, more specifically alkalinization, affect the apoptotic cascade has yet to be determined. The aim of the present work was to test the involvement of mitochondria in the apoptotic cascade triggered by benzo(a)pyrene [B(a)P] and to determine the role of pH(i) changes and p53 relative to mitochondria. Our results indicate that B(a)P-induced apoptosis might rely upon a p53-dependent and a pH-sensitive mitochondrial dysfunction.

  6. Synthesis and anticancer potential of benzothiazole linked phenylpyridopyrimidinones and their diones as mitochondrial apoptotic inducers.

    PubMed

    Kamal, Ahmed; Ashraf, Md; Vishnu Vardhan, M V P S; Faazil, Shaikh; Nayak, V Lakshma

    2014-01-01

    A series of benzothiazole linked phenylpyridopyrimidinones (8a-g) and their diones (9a-g) have been designed, synthesized and evaluated for their anticancer activity. Among the series one of the conjugate 8b showed significant cytotoxicity against human cervical cancer cell line ME-180 with IC50 value of 4.01μM. This compound was tested on the cell cycle perturbations and DNA damage. Flow cytometry analysis revealed that the compound 8b showed drastic cell cycle perturbations due to concentration dependent increase in the sub-G0 phase in ME-180 cell line. DNA fragmentation and Hoechst staining reveals that this compound induced cell death by apoptosis. Further caspase-3 and loss of mitochondrial membrane potential suggested that the compound induces cell death by apoptosis.

  7. Galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-Bid mitochondrial pathway.

    PubMed

    Zhang, Hai-Tao; Wu, Jun; Wen, Min; Su, Li-Juan; Luo, Hui

    2012-01-01

    This study has investigated whether galangin, a flavonol derived from Alpinia officinarum Hance and used as food additives in southern China, induces apoptosis in hepatocellular carcinoma cells (HCCs) by activation of the caspase-8 and Bid pathway. The apoptosis of HCCs was evaluated by in situ uptake of propidium iodide and Hoechst 33258. Protein expressions were detected by Western blotting. Caspase-8 activity was measured using colorimetric method. To confirm the galangin-induced apoptotic pathway, inhibition of caspase-8 activity by Z-IETD-FMK, knockdown of Bid expression with siRNA, and overexpression of Bcl-2 in cells were carried out, respectively. The results show that galangin has significantly induced apoptosis in HCC lines. The caspase-8 is activated, and the cleavage of Bid results in the increase in tBid. The galangin-induced apoptosis is attenuated by Z-IETD-FMK, Bid siRNA, and Bcl-2 overexpression, respectively. However, Bcl-2 fails to suppress caspase-8 activation and the cleavage of Bid. This study has demonstrated that galangin induces apoptosis in HCCs by activating caspase 8/t-Bid mitochondrial pathway. Although Bcl-2 overexpression attenuates galangin-mediated apoptosis of HCCs, it is not mediated by the inhibition of tBid generation and caspase-8 activation.

  8. Tricholoma matsutake Aqueous Extract Induces Hepatocellular Carcinoma Cell Apoptosis via Caspase-Dependent Mitochondrial Pathway

    PubMed Central

    Wang, Yanzhen; Chen, Yiling; Zhang, Xinrui; Cai, Guangsheng; An, Shengshu; Wang, Xue

    2016-01-01

    Tricholoma matsutake, one of widely accepted functional mushrooms, possesses various pharmacological activities, and its antitumor effect has become an important research point. Our study aims to evaluate the cytotoxicity activities of T. matsutake aqueous extract (TM) in HepG2 and SMMC-7721 cells. In in vitro experiments, TM strikingly reduced cell viability, promoted cell apoptosis, inhibited cell migration ability, induced excessive generation of ROS, and caused caspases cascade and mitochondrial membrane potential dissipation in hepatocellular carcinoma cells. In in vivo experiments, 14-day TM treatment strongly suppressed tumor growth in HepG2 and SMMC-7721-xenografted nude mice without influence on their body weights and liver function. Furthermore, TM increased the levels of cleaved poly-ADP-ribose polymerase (PARP), Bad, and Bax and reduced the expressions of B-cell lymphoma 2 (Bcl-2) in treated cells and tumor tissues. All aforementioned results suggest that caspase-dependent mitochondrial apoptotic pathways are involved in TM-mediated antihepatocellular carcinoma. PMID:28018916

  9. Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway.

    PubMed

    Wang, Xi; Lu, Xiaocheng; Zhu, Ronglan; Zhang, Kaixin; Li, Shuai; Chen, Zhongjun; Li, Lixin

    2017-01-25

    Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

  10. Xanthohumol induces different cytotoxicity and apoptotic pathways in malignant and normal astrocytes.

    PubMed

    Zajc, I; Filipič, M; Lah, T T

    2012-11-01

    Cytotoxicity and the mechanisms of cell death induced by xanthohumol (XN) were compared in normal and cancerous human cells as the differences may be relevant for the potential use of XN in cancer therapy. The cancer cells seemed to be more susceptible to the cytotoxicity of XN than normal cells, but a significant difference was observed only in astrocytic cells. XN induced a higher rate of apoptosis in glioblastoma cells than in normal astrocytes, which was associated with activation of p53 and an elevated Bax/Bcl-2 ratio in glioblastoma cells, indicating an intrinsic caspase-dependent apoptotic pathway. In contrast, a reduced Bax/Bcl-2 ratio was observed in normal human astrocytes. This was also associated with higher expression of the cell cycle inhibitor, p21, in glioblastoma cells than in normal astrocytes. In addition, at a lower, non-cytotoxic concentration, XN partially inhibited the invasiveness of glioblastoma cells. Due to the selective sensitivity of astrocytic cells to XN, this compound should be studied further as a candidate for adjuvant therapy in the treatment of glioma.

  11. Insights into sepsis therapeutic design based on the apoptotic death pathway.

    PubMed

    Hattori, Yuichi; Takano, Ken-ichi; Teramae, Hiroki; Yamamoto, Seiji; Yokoo, Hiroki; Matsuda, Naoyuki

    2010-01-01

    Sepsis remains the leading cause of death in critically ill patients. A major problem contributing to sepsis-related high mortality is the lack of effective medical treatment. Thus, the key goal in critical care medicine is to develop novel therapeutic strategies that will impact favorably on septic patient outcome. While it is generally accepted that sepsis is an inflammatory state resulting from the systemic response to infection, apoptosis is implicated to be an important mechanism of the death of lymphocytes, gastrointestinal and lung epithelial cells, and vascular endothelial cells associated with the development of multiple organ failure in sepsis. The pivotal role of cell apoptosis is now highlighted by multiple studies demonstrating that prevention of cell apoptosis can improve survival in clinically relevant animal models of sepsis. In this review article, we address the scientific rationale for remedying apoptotic cell death in sepsis and propose that therapeutic efforts aimed at blocking cell signaling pathways leading to apoptosis may represent an attractive target for sepsis therapy.

  12. Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma.

    PubMed

    Syed, Deeba N; Lall, Rahul K; Chamcheu, Jean Christopher; Haidar, Omar; Mukhtar, Hasan

    2014-12-01

    The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytotoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up-regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMP-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity.

  13. A neuronal C5a receptor and an associated apoptotic signal transduction pathway.

    PubMed

    Farkas, I; Baranyi, L; Takahashi, M; Fukuda, A; Liposits, Z; Yamamoto, T; Okada, H

    1998-03-15

    1. We report the first experimental evidence of a neuronal C5a receptor (nC5aR) in human cells of neuronal origin. Expression of nC5aR mRNA was demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) in TGW human neuroblastoma cells. 2. Expression of a functional C5aR was supported by the finding that C5a evoked a transient increase in the intracellular calcium level as measured by flow cytometry (FACS). 3. To analyse the function of the nC5aR, an antisense peptide fragment of the C5aR was used. Previous data showed that a C5aR fragment (a peptide termed PR226) has C5aR agonist and antagonist effects in U-937 cells depending on the concentration of the peptide. We found that a multiple antigenic peptide (MAP) form of the same peptide (termed PR226-MAP) induced rapid elevation of nuclear c-fos immunoreactivity and resulted in DNA fragmentation, a characteristic sign of apoptosis, in TGW cells. 4. Early electrophysiological events characteristic of apoptosis were also detected: intermittent calcium current pulses were recorded within 1-2 min of peptide administration. C5a pretreatment delayed the onset of this calcium influx. 5. We also demonstrated that the apoptotic pathway is linked to nC5aR via pertussis toxin-sensitive G-proteins. 6. Although the function of C5a and its receptor on neurons is unknown, these results suggest that an abnormal activation of this signal transduction pathway can result in apoptosis and, subsequently, in neurodegeneration.

  14. The Macrophage Phagocytic Receptor CD36 Promotes Fibrogenic Pathways on Removal of Apoptotic Cells during Chronic Kidney Injury

    PubMed Central

    Pennathur, Subramaniam; Pasichnyk, Katie; Bahrami, Nadia M.; Zeng, Lixia; Febbraio, Maria; Yamaguchi, Ikuyo; Okamura, Daryl M.

    2016-01-01

    The removal of apoptotic cells is an innate function of tissue macrophages; however, its role in disease progression is unclear. The present study was designed to investigate the role of macrophage CD36, a recognized receptor of apoptotic cells and oxidized lipids, in two models of kidney injury: unilateral ureteral obstruction (UUO) and ischemia reperfusion. To differentiate the macrophage CD36-specific effects in vivo, we generated CD36 chimeric mice by bone marrow transplantation and evaluated the two models. Fibrosis severity was substantially decreased after UUO with a corresponding decrease in matrix synthesis in macrophage CD36-deficient mice. Despite a reduction in fibrosis severity, a 56% increase in apoptotic cells was found without an increase in apoptotic effectors. In addition, a substantial reduction was observed in tumor necrosis factor-α and transforming growth factor-β1 mRNA levels and intracellular bioactive oxidized lipid levels in CD36-deficient macrophages. To validate the functional role of macrophage CD36, we performed unilateral ischemia reperfusion, followed by contralateral nephrectomy. Similarly, we found that the severity of fibrosis was reduced by 55% with a corresponding improvement in kidney function by 88% in macrophage CD36-deficient mice. Taken together, these data suggest that macrophage CD36 is a critical regulator of oxidative fibrogenic signaling and that CD36-mediated phagocytosis of apoptotic cells may serve as an important pathway in the progression of fibrosis. PMID:26092500

  15. BL-038, a Benzofuran Derivative, Induces Cell Apoptosis in Human Chondrosarcoma Cells through Reactive Oxygen Species/Mitochondrial Dysfunction and the Caspases Dependent Pathway.

    PubMed

    Liu, Ju-Fang; Chen, Chien-Yu; Chen, Hsien-Te; Chang, Chih-Shiang; Tang, Chih-Hsin

    2016-09-07

    Chondrosarcoma is a highly malignant cartilage-forming bone tumor that has the capacity to invade locally and cause distant metastasis. Moreover, chondrosarcoma is intrinsically resistant to conventional chemotherapy or radiotherapy. The novel benzofuran derivative, BL-038 (2-amino-3-(2,6-dichlorophenyl)-6-(4-methoxyphenyl)benzofuran-4-yl acetate), has been evaluated for its anticancer effects in human chondrosarcoma cells. BL-038 caused cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353, but not in primary chondrocytes. Treatment of chondrosarcoma with BL-038 also induced reactive oxygen species (ROS) production. Furthermore, BL-038 decreased mitochondrial membrane potential (MMP) and changed mitochondrial-related apoptosis, by downregulating the anti-apoptotic activity members (Bcl-2, Bcl-xL) and upregulating pro-apoptotic members (Bax, Bak) of the B-cell lymphoma 2 (Bcl-2) family of proteins, key regulators of the apoptotic machinery in cells. These results demonstrate that in human chondrosarcoma cells, the apoptotic and cytotoxic effects of BL-038 are mediated by the intrinsic mitochondria-mediated apoptotic pathway, which in turn causes the release of cytochrome c, the activation of caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP), to elicit apoptosis response. Our results show that the benzofuran derivative BL-038 induces apoptosis in chondrosarcoma cells.

  16. MiADMSA reverses impaired mitochondrial energy metabolism and neuronal apoptotic cell death after arsenic exposure in rats

    SciTech Connect

    Dwivedi, Nidhi; Mehta, Ashish; Yadav, Abhishek; Binukumar, B.K.; Gill, Kiran Dip; Flora, Swaran J.S.

    2011-11-15

    Arsenicosis, due to contaminated drinking water, is a serious health hazard in terms of morbidity and mortality. Arsenic induced free radicals generated are known to cause cellular apoptosis through mitochondrial driven pathway. In the present study, we investigated the effect of arsenic interactions with various complexes of the electron transport chain and attempted to evaluate if there was any complex preference of arsenic that could trigger apoptosis. We also evaluated if chelation with monoisoamyl dimercaptosuccinic acid (MiADMSA) could reverse these detrimental effects. Our results indicate that arsenic exposure induced free radical generation in rat neuronal cells, which diminished mitochondrial potential and enzyme activities of all the complexes of the electron transport chain. Moreover, these complexes showed differential responses towards arsenic. These early events along with diminished ATP levels could be co-related with the later events of cytosolic migration of cytochrome c, altered bax/bcl{sub 2} ratio, and increased caspase 3 activity. Although MiADMSA could reverse most of these arsenic-induced altered variables to various extents, DNA damage remained unaffected. Our study for the first time demonstrates the differential effect of arsenic on the complexes leading to deficits in bioenergetics leading to apoptosis in rat brain. However, more in depth studies are warranted for better understanding of arsenic interactions with the mitochondria. -- Research highlights: Black-Right-Pointing-Pointer Arsenic impairs mitochondrial energy metabolism leading to neuronal apoptosis. Black-Right-Pointing-Pointer Arsenic differentially affects mitochondrial complexes, I - III and IV being more sensitive than complex II. Black-Right-Pointing-Pointer Arsenic-induced apoptosis initiates through ROS generation or impaired [Ca{sup 2+}]i homeostasis. Black-Right-Pointing-Pointer MiADMSA reverses arsenic toxicity via intracellular arsenic- chelation, antioxidant

  17. Lithospermic acid B protects beta-cells from cytokine-induced apoptosis by alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1

    SciTech Connect

    Lee, Byung-Wan; Chun, Sung Wan; Kim, Soo Hyun; Lee, Yongho; Kang, Eun Seok; Cha, Bong-Soo; Lee, Hyun Chul

    2011-04-01

    Lithospermic acid B (LAB) has been reported to protect OLETF rats, an established type 2 diabetic animal model, from the development of diabetes-related vascular complications. We investigated whether magnesium lithospermate B (LAB) has a protective role under cytokine-induced apoptosis in INS-1 cells in vitro and whether it slows the development of diabetes in OLETF rats in vivo. Pretreatment with 50 {mu}M LAB significantly reduced the 1000 U/mL INF-{gamma} and 100 U/mL IL-1{beta}-induced INS-1 cell death. LAB significantly alleviated cytokine-induced phosphorylations of p38 and JNK in accordance with a decrease in cleaved caspase-3 activity in beta-cells. LAB also protected against the cytokine-induced caspase-3 apoptotic pathway via significant activation of Nrf2-HO (heme-oxigenase)-1 and Sirt1 expression. OLETF rats treated with 40 mg/kg/day LAB showed a significant improvement in glucose tolerance compared to untreated OLETF control rats in vivo. Our results suggest that the cytoprotective effects of LAB on pancreatic {beta}-cells are related with both alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1.

  18. Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

    PubMed Central

    Hsiao, Yu-Ping; Lai, Wan-Wen; Wu, Shi-Bei; Tsai, Chung-Hung; Tang, Sheau-Chung; Chung, Jing-Gung; Yang, Jen-Hung

    2015-01-01

    Malic acid (MA) has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT). The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs). Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX) but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR) was significantly decreased whereas extracellular acidification rate (ECAR) was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways. PMID:25584429

  19. Diallyl disulfide attenuated carbon ion irradiation-induced apoptosis in mouse testis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway.

    PubMed

    Di, Cui-xia; Han, Lu; Zhang, Hong; Xu, Shuai; Mao, Ai-hong; Sun, Chao; Liu, Yang; Si, Jing; Li, Hong-yan; Zhou, Xin; Liu, Bing; Miao, Guo-ying

    2015-11-03

    Diallyl disulfide (DADS), a major organosulfur compound derived from garlic, has various biological properties, including anti-cancer effects. However, the protective mechanism of DADS against radiation-induced mouse testis cell apoptosis has not been elucidated. In this study, the magnitude of radiation effects evoked by carbon ion irradiation was marked by morphology changes, significant rise in apoptotic cells, activation expression of p53, up regulation the ratio of pro-apoptotic Tap73/anti-apoptotic ΔNp73, as well as alterations of crucial mediator of the mitochondrial pathway. Interestingly, pretreatment with DADS attenuated carbon ion irradiation-induced morphology damages and apoptotic cells. Additionally, DADS elevated radiation-induced p53 and p21 expression, suggesting that p53 might be involved in the inhibition of cell cycle progression through up regulation of p21. Furthermore, administration with DADS prevented radiation-induced Tap73/ΔNp73 expression and consequently down regulated Bax/Bcl-2 ratio, cytochrome c release and caspase-3 expression, indicating that the balance between Tap73 and ΔNp73 had potential to activate p53 responsive genes. Thus, our results showed that radio protection effect of DADS on mouse testis is mediated by blocking apoptosis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway, suggesting that DADS could be used as a potential radio protection agent for the testis against heavy-ion radiation.

  20. Diallyl disulfide attenuated carbon ion irradiation-induced apoptosis in mouse testis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway

    PubMed Central

    Di, Cui-xia; Han, Lu; Zhang, Hong; Xu, Shuai; Mao, Ai-hong; Sun, Chao; Liu, Yang; Si, Jing; Li, Hong-yan; Zhou, Xin; Liu, Bing; Miao, Guo-ying

    2015-01-01

    Diallyl disulfide (DADS), a major organosulfur compound derived from garlic, has various biological properties, including anti-cancer effects. However, the protective mechanism of DADS against radiation-induced mouse testis cell apoptosis has not been elucidated. In this study, the magnitude of radiation effects evoked by carbon ion irradiation was marked by morphology changes, significant rise in apoptotic cells, activation expression of p53, up regulation the ratio of pro-apoptotic Tap73/anti-apoptotic ΔNp73, as well as alterations of crucial mediator of the mitochondrial pathway. Interestingly, pretreatment with DADS attenuated carbon ion irradiation-induced morphology damages and apoptotic cells. Additionally, DADS elevated radiation-induced p53 and p21 expression, suggesting that p53 might be involved in the inhibition of cell cycle progression through up regulation of p21. Furthermore, administration with DADS prevented radiation-induced Tap73/ΔNp73 expression and consequently down regulated Bax/Bcl-2 ratio, cytochrome c release and caspase-3 expression, indicating that the balance between Tap73 and ΔNp73 had potential to activate p53 responsive genes. Thus, our results showed that radio protection effect of DADS on mouse testis is mediated by blocking apoptosis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway, suggesting that DADS could be used as a potential radio protection agent for the testis against heavy-ion radiation. PMID:26526304

  1. Mitochondrial dysfunction associated with nitric oxide pathways in glutamate neurotoxicity.

    PubMed

    Manucha, Walter

    Multiple mechanisms underlying glutamate-induced neurotoxicity have recently been discussed. Likewise, a clear deregulation of the mitochondrial respiratory mechanism has been described in patients with neurodegeneration, oxidative stress, and inflammation. This article highlights nitric oxide, an atypical neurotransmitter synthesized and released on demand by the post-synaptic neurons, and has many important implications for nerve cell survival and differentiation. Consequently, synaptogenesis, synapse elimination, and neurotransmitter release, are nitric oxide-modulated. Interesting, an emergent role of nitric oxide pathways has been discussed as regards neurotoxicity from glutamate-induced apoptosis. These findings suggest that nitric oxide pathways modulation could prevent oxidative damage to neurons through apoptosis inhibition. This review aims to highlight the emergent aspects of nitric oxide-mediated signaling in the brain, and how they can be related to neurotoxicity, as well as the development of neurodegenerative diseases development.

  2. Deoxynivalenol induces apoptosis in PC12 cells via the mitochondrial pathway.

    PubMed

    Wang, Xichun; Xu, Wei; Fan, Mengxue; Meng, Tingting; Chen, Xiaofang; Jiang, Yunjing; Zhu, Dianfeng; Hu, Wenjuan; Gong, Jiajie; Feng, Shibin; Wu, Jinjie; Li, Yu

    2016-04-01

    Deoxynivalenol (DON) has broad toxicity in animals and humans. In this study the impact of DON treatment on apoptotic pathways in PC12 cells was determined. The effects of DON were evaluated on (i) typical indicators of apoptosis, including cellular morphology, cell activity, lactate dehydrogenase (LDH) release, and apoptosis ratio in PC12 cells, and on (ii) the expression of key genes and proteins related to apoptosis, including Bcl-2, Bax, Bid, cytochrome C (Cyt C), apoptosis inducing factor (AIF), cleaved-Caspase9, and cleaved-Caspase3. DON treatment inhibited proliferation of PC12 cells, induced significant morphological changes and apoptosis, promoted the release of Cyt C and AIF from the mitochondria, and increased the activities of cleaved-Caspase9 and cleaved-Caspase3. Bcl-2 expression decreased with increasing DON concentrations, in contrast to Bax and Bid, which were increased with increasing DON concentration. These data demonstrate that DON induces apoptosis in PC12 cells through the mitochondrial apoptosis pathway.

  3. Cardiac Fas-Dependent and Mitochondria-Dependent Apoptotic Pathways in a Transgenic Mouse Model of Huntington's Disease.

    PubMed

    Wu, Bor-Tsang; Chiang, Ming-Chang; Tasi, Ching-Yi; Kuo, Chia-Hua; Shyu, Woei-Cherng; Kao, Chung-Lan; Huang, Chih-Yang; Lee, Shin-Da

    2016-04-01

    Huntington's disease is an autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in the huntingtin gene. Heart disease is the second leading cause of death in patients with Huntington's disease. This study was to evaluate whether cardiac Fas-dependent and mitochondria-dependent apoptotic pathways are activated in transgenic mice with Huntington's disease. Sixteen Huntington's disease transgenic mice (HD) and sixteen wild-type (WT) littermates were studied at 10.5 weeks of age. The cardiac characteristics, myocardial architecture, and two major apoptotic pathways in the excised left ventricle from mice were measured by histopathological analysis, Western blotting, and TUNEL assays. The whole heart weight and the left ventricular weight decreased significantly in the HD group, as compared to the WT group. Abnormal myocardial architecture, enlarged interstitial spaces, and more cardiac TUNEL-positive cells were observed in the HD group. The key components of Fas-dependent apoptosis (TNF-alpha, TNFR1, Fas ligand, Fas death receptors, FADD, activated caspase-8, and activated caspase-3) and the key components of mitochondria-dependent apoptosis (Bax, Bax-to-Bcl-2 ratio, cytosolic cytochrome c, activated caspase-9, and activated caspase-3) increased significantly in the hearts of the HD group. Cardiac Fas-dependent and mitochondria-dependent apoptotic pathways were activated in transgenic mice with Huntington's disease, which might provide one of possible mechanisms to explain why patients with Huntington's disease will develop heart failure.

  4. Inorganic mercury causes pancreatic beta-cell death via the oxidative stress-induced apoptotic and necrotic pathways

    SciTech Connect

    Chen Yawen; Huang Chunfa; Yang Chingyao; Yen Chengchieh; Tsai Kehsung; Liu Shinghwa

    2010-03-15

    Mercury is a well-known highly toxic metal. In this study, we characterize and investigate the cytotoxicity and its possible mechanisms of inorganic mercury in pancreatic beta-cells. Mercury chloride (HgCl{sub 2}) dose-dependently decreased the function of insulin secretion and cell viability in pancreatic beta-cell-derived HIT-T15 cells and isolated mouse pancreatic islets. HgCl{sub 2} significantly increased ROS formation in HIT-T15 cells. Antioxidant N-acetylcysteine effectively reversed HgCl{sub 2}-induced insulin secretion dysfunction in HIT-T15 cells and isolated mouse pancreatic islets. Moreover, HgCl{sub 2} increased sub-G1 hypodiploids and annexin-V binding in HIT-T15 cells, indicating that HgCl{sub 2} possessed ability in apoptosis induction. HgCl{sub 2} also displayed several features of mitochondria-dependent apoptotic signals including disruption of the mitochondrial membrane potential, increase of mitochondrial cytochrome c release and activations of poly (ADP-ribose) polymerase (PARP) and caspase 3. Exposure of HIT-T15 cells to HgCl{sub 2} could significantly increase both apoptotic and necrotic cell populations by acridine orange/ethidium bromide dual staining. Meanwhile, HgCl{sub 2} could also trigger the depletion of intracellular ATP levels and increase the LDH release from HIT-T15 cells. These HgCl{sub 2}-induced cell death-related signals could be significantly reversed by N-acetylcysteine. The intracellular mercury levels were markedly elevated in HgCl{sub 2}-treated HIT-T15 cells. Taken together, these results suggest that HgCl{sub 2}-induced oxidative stress causes pancreatic beta-cell dysfunction and cytotoxicity involved the co-existence of apoptotic and necrotic cell death.

  5. The Effect of Selenium on the Cd-Induced Apoptosis via NO-Mediated Mitochondrial Apoptosis Pathway in Chicken Liver.

    PubMed

    Zhang, Runxiang; Yi, Ran; Bi, Yanju; Xing, Lu; Bao, Jun; Li, Jianhong

    2017-01-06

    Cd-induced apoptosis and the protective effects of Se against Cd-induced injury have been reported in previous studies. However, little is known regarding the effects of Cd-induced apoptosis in hepatic cells and the antagonistic effects of Se on Cd in poultry. In the present study, 128 healthy 31-week-old laying hens were randomly divided into four groups, which were fed basic diets, with the addition of Se (Na2SeO3, 2 mg/kg), Cd (CdCl2, 150 mg/kg), or Se + Cd (150 mg/kg of CdCl2 and 2 mg/kg of Na2SeO3) for 90 days. Ultrastructural changes, nitric oxide (NO) concentrations, inducible nitric oxide synthase (iNOS) activities, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and the expression of iNOS and apoptosis-related genes in livers were determined. It was observed that Cd treatment significantly increased the concentrations of NO and iNOS activity in chicken livers. The production of excessive NO initiated the mitochondrial apoptotic pathway. Exposure to Cd increased the mRNA and the protein expression levels of iNOS, caspase-3, Bax, p53, and Cyt-c. Furthermore, the ratio of Bax/Bcl-2 increased, while the expression of Bcl-2 decreased. Treatment with Se significantly alleviated Cd-induced apoptosis in chicken livers, as evidenced by a reduction in the production of NO, iNOS activity, the number of apoptotic cells, and mRNA and protein expression levels of iNOS, caspase-3, Bax, and Cyt-c. It indicated that Cd induced NO-mediated apoptosis through the mitochondrial apoptotic pathway and Se exerted antagonizing effects. The present study provides new insights as to how Se affects Cd-induced toxicity in the chicken liver.

  6. Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion*

    PubMed Central

    Catalán, Elena; Jaime-Sánchez, Paula; Aguiló, Nacho; Simon, Markus M.; Froelich, Christopher J.; Pardo, Julián

    2015-01-01

    Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB+Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB+Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB+Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB+Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB+Tc-induced death pathways. PMID:25605735

  7. Mouse cytotoxic T cell-derived granzyme B activates the mitochondrial cell death pathway in a Bim-dependent fashion.

    PubMed

    Catalán, Elena; Jaime-Sánchez, Paula; Aguiló, Nacho; Simon, Markus M; Froelich, Christopher J; Pardo, Julián

    2015-03-13

    Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB(+)Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB(+)Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB(+)Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB(+)Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB(+)Tc-induced death pathways.

  8. Microcystin-LR Induced Apoptosis in Rat Sertoli Cells via the Mitochondrial Caspase-Dependent Pathway: Role of Reactive Oxygen Species

    PubMed Central

    Huang, Hui; Liu, Chuanrui; Fu, Xiaoli; Zhang, Shenshen; Xin, Yongjuan; Li, Yang; Xue, Lijian; Cheng, Xuemin; Zhang, Huizhen

    2016-01-01

    Microcystins (MCs), the secondary metabolites of blue-green algae, are ubiquitous and major cyanotoxin contaminants. Besides the hepatopancreas/liver, the reproductive system is regarded as the most important target organ for MCs. Although reactive oxygen species (ROS) have been implicated in MCs-induced reproductive toxicity, the role of MCs in this pathway remains unclear. In the present study, Sertoli cells were employed to investigate apoptotic death involved in male reproductive toxicity of microcystin-LR (MC-LR). After exposure to various concentrations of MC-LR for 24 h, the growth of Sertoli cells was concentration-dependently decreased with an IC50 of ~32 μg/mL. Mitochondria-mediated apoptotic changes were observed in Sertoli cells exposed to 8, 16, and 32 μg/mL MC-LR including the increased expression of caspase pathway proteins, collapse of mitochondrial membrane potential (MMP), and generation of ROS. Pretreatment with a global caspase inhibitor was found to depress the activation of caspases, and eventually increased the survival rate of Sertoli cells, implying that the mitochondrial caspases pathway is involved in MC-LR-induced apoptosis. Furthermore, N-acetyl-l-cysteine attenuated the MC-LR-induced intracellular ROS generation, MMP collapse and cytochrome c release, resulting in the inhibition of apoptosis. Taken together, the observed results suggested that MC-LR induced apoptotic death of Sertoli cells by the activation of mitochondrial caspases cascade, while its effects on the ROS-mediated signaling pathway may contribute toward the initiation of mitochondrial dysfunction. PMID:27667976

  9. Phellinus linteus polysaccharide extracts increase the mitochondrial membrane potential and cause apoptotic death of THP-1 monocytes

    PubMed Central

    2013-01-01

    Background The differentiation resp. death of human monocytic THP-1 cells induced by polysaccharide extracts of the medicinal mushrooms Phellinus linteus, Agaricus bisporus and Agaricus brasiliensis have been studied. This study aims to identify leads for the causal effects of these mushroom components on cell differentiation and death. Methods THP-1 cells were treated with different polysaccharide extracts of mushrooms and controls. Morphological effects were observed by light microscopy. Flow cytometry was applied to follow the cell differentiation by cell cycle shifts after staining with propidium iodide, changes of mitochondrial membrane potential after incubation with JC-1, and occurrence of intracellular reactive oxygen species after incubation with hydroethidine. Principal component analysis of the data was performed to evaluate the cellular effects of the different treatments. Results P. linteus polysaccharide extracts induced dose-dependent apoptosis of THP-1 cells within 24 h, while A. bisporus and A. brasiliensis polysaccharide extracts caused differentiation into macrophages. A pure P. linteus polysaccharide had no effect. Apoptosis was inhibited by preincubating THP-1 cells with human serum. The principal component analysis revealed that P. linteus, A. bisporus and A. brasiliensis polysaccharide extracts increased reactive oxygen species production. Both A. bisporus and A. brasiliensis polysaccharide extracts decreased the mitochondrial membrane potential, while this was increased by P. linteus polysaccharide extracts. Conclusions P. linteus polysaccharide extracts caused apoptosis of THP-1 monocytes while A. bisporus and A. brasiliensis polysaccharide extracts caused these cells to differentiate into macrophages. The protective effects of human serum suggested that P. linteus polysaccharide extract induced apoptosis by extrinsic pathway, i.e. by binding to the TRAIL receptor. The mitochondrial membrane potential together with reactive oxygen species

  10. Role of SIRT1-mediated mitochondrial and Akt pathways in glioblastoma cell death induced by Cotinus coggygria flavonoid nanoliposomes

    PubMed Central

    Wang, Gang; Wang, Jun Jie; To, Tony SS; Zhao, Hua Fu; Wang, Jing

    2015-01-01

    Flavonoids, the major polyphenol components in Cotinus coggygria (CC), have been found to show an anticancer effect in our previous study; however, the exact mechanisms of inducing human glioblastoma (GBM) cell death remain to be resolved. In this study, a novel polyvinylpyrrolidone K-30/sodium dodecyl sulfate and polyethyleneglycol-coated liposome loaded with CC flavonoids (CCFs) was developed to enhance solubility and the antibrain tumor effect, and the molecular mechanism regarding how CCF nanoliposomes (CCF-NLs) induce apoptotic cell death in vitro was investigated. DBTRG-05MG GBM cell lines treated with CCF-NLs showed potential antiproliferative effects. Regarding the underlying mechanisms of inducing apoptosis in DBTRG-05MG GBM cells, CCF-NLs were shown to downregulate the expression of antiapoptotic B-cell lymphoma/leukemia 2 (Bcl-2), an apoptosis-related protein family member, but the expression of proapoptotic Bcl-2-associated X protein was enhanced compared with that in controls. CCF-NLs also inhibited the activity of caspase-3 and -9, which is the initiator caspase of the extrinsic and intrinsic apoptotic pathways. Blockade of caspase activation consistently induced apoptosis and inhibited growth in CCF-NL-treated DBTRG-05MG cells. This study further investigated the role of the Akt pathway in the apoptotic cell death by CCF-NLs, showing that CCF-NLs deactivated Akt. Specifically, CCF-NLs downregulated the expression of p-Akt and SIRT1 as well as the level of phosphorylated p53. Together, these results indicated SIRT1/p53-mediated cell death was induced by CCF-NLs, but not by extracellular signal-regulated kinase, in DBTRG-05MG cells. Overall, this study suggested caspase-dependent activation of both the intrinsic and extrinsic signaling pathways, probably through blockade of the SIRT1/p53-mediated mitochondrial and Akt pathways to exert the proapoptotic effect of CCF-NLs in DBTRG-05MG GBM cells. PMID:26345416

  11. A polysaccharide from Glycyrrhiza inflata Licorice inhibits proliferation of human oral cancer cells by inducing apoptosis via mitochondrial pathway.

    PubMed

    Shen, Huan; Zeng, Guang; Sun, Bin; Cai, Xingwei; Bi, Lixia; Tang, Guo; Yang, Yongjin

    2015-06-01

    In the present study, we isolated and characterized a water-soluble polysaccharide (GIP1) from the roots of Glycyrrhiza inflata. The goal of this study was to investigate the anti-tumor effect of GIP1 on the human oral cancer SCC-25 cell line and to explore the possible mechanism. Our experimental result showed that GIP1 (50, 100, and 200 μg/mL) specifically decreased cell viability of SCC-25 cells in a concentration-dependent manner via the induction of apoptosis. Furthermore, Western blot analysis showed that exposure of SCC-25 cells to GIP1 led to down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, thus causing a loss of mitochondrial membrane potential and the release of cytochrome c to the cytosol. Moreover, we observed activation of the initiator caspaes-9, and the effector caspases-3, but not caspase-8. Concomitantly, GIP1-induced apoptosis can be blocked by caspase-3- or caspase-9-specific inhibitor, but not caspase-8 inhibitor. As well, the cleaved poly (ADP-ribose) polymerase, as a caspae-3 substrate, occurred in SCC-25 cells following GIP1 treatment at three concentrations. Collectively, our results showed that the GIP1 induced apoptosis in SCC-25 cells involving a caspase-dependent mitochondrial signaling pathway.

  12. Coenzyme Q10 Ameliorates Ultraviolet B Irradiation Induced Cell Death Through Inhibition of Mitochondrial Intrinsic Cell Death Pathway

    PubMed Central

    Jing, Li; Kumari, Santosh; Mendelev, Natalia; Li, P. Andy

    2011-01-01

    Ultraviolet B (UVB) induces cell death by increasing free radical production, activating apoptotic cell death pathways and depolarizing mitochondrial membrane potential. Coenzyme Q10 (CoQ10), an essential cofactor in the mitochondrial electron transport chain, serves as a potent antioxidant in the mitochondria. The aim of the present study is to establish whether CoQ10 is capable of protecting neuronal cells against UVB-induced damage. Murine hippocampal HT22 cells were treated with 0.01, 0.1 or 1 μM of CoQ10 3 or 24 h prior to the cells being exposed to UVB irradiation. The CoQ10 concentrations were maintained during irradiation and 24 h post-UVB. Cell viability was assessed by counting viable cells and MTT conversion assay. Superoxide production and mitochondrial membrane potential were measured using fluorescent probes. Levels of cleaved caspase-9, caspase-3, and apoptosis-inducing factor (AIF) were detected using immunocytochemistry and Western blotting. The results showed that UVB irradiation decreased cell viability and such damaging effect was associated with increased superoxide production, mitochondrial depolarization, and activation of caspase-9 and caspase-3. Treatment with CoQ10 at three different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide production, normalization of mitochondrial membrane potential and inhibition of caspase-9 and caspase-3 activation. It is concluded that the neuroprotective effect of CoQ10 results from inhibiting oxidative stress and blocking caspase-3 dependent cell death pathway. PMID:22174665

  13. AS-2, a novel inhibitor of p53-dependent apoptosis, prevents apoptotic mitochondrial dysfunction in a transcription-independent manner and protects mice from a lethal dose of ionizing radiation

    SciTech Connect

    Morita, Akinori; Ariyasu, Shinya; Wang, Bing; Asanuma, Tetsuo; Onoda, Takayoshi; Sawa, Akiko; Tanaka, Kaoru; Takahashi, Ippei; Togami, Shotaro; Nenoi, Mitsuru; Inaba, Toshiya; Aoki, Shin

    2014-08-08

    Highlights: • A bidentate HQ derivative, AS-2, suppresses p53-dependent apoptosis by DNA damage. • AS-2 does not significantly affect nuclear p53 response. • UV-excited blue emission of AS-2 clearly showed its extranuclear localization. • AS-2 prevents mitochondrial dysfunction despite the increase of mitochondrial p53. • AS-2 protects mice from a radiation dose that causes lethal hematopoietic syndrome. - Abstract: In a previous study, we reported that some tetradentate zinc(II) chelators inhibit p53 through the denaturation of its zinc-requiring structure but a chelator, Bispicen, a potent inhibitor of in vitro apoptosis, failed to show any efficient radioprotective effect against irradiated mice because the toxicity of the chelator to mice. The unsuitability of using tetradentate chelators as radioprotectors prompted us to undertake a more extensive search for p53-inhibiting agents that are weaker zinc(II) chelators and therefore less toxic. Here, we show that an 8-hydroxyquinoline (8HQ) derivative, AS-2, suppresses p53-dependent apoptosis through a transcription-independent mechanism. A mechanistic study using cells with different p53 characteristics revealed that the suppressive effect of AS-2 on apoptosis is specifically mediated through p53. In addition, AS-2 was less effective in preventing p53-mediated transcription-dependent events than pifithrin-μ (PFTμ), an inhibitor of transcription-independent apoptosis by p53. Fluorescence visualization of the extranuclear distribution of AS-2 also supports that it is ineffective on the transcription-dependent pathway. Further investigations revealed that AS-2 suppressed mitochondrial apoptotic events, such as the mitochondrial release of intermembrane proteins and the loss of mitochondrial membrane potential, although AS-2 resulted in an increase in the mitochondrial translocation of p53 as opposed to the decrease of cytosolic p53, and did not affect the apoptotic interaction of p53 with Bcl-2. AS-2 also

  14. Circadian acetylome reveals regulation of mitochondrial metabolic pathways.

    PubMed

    Masri, Selma; Patel, Vishal R; Eckel-Mahan, Kristin L; Peleg, Shahaf; Forne, Ignasi; Ladurner, Andreas G; Baldi, Pierre; Imhof, Axel; Sassone-Corsi, Paolo

    2013-02-26

    The circadian clock is constituted by a complex molecular network that integrates a number of regulatory cues needed to maintain organismal homeostasis. To this effect, posttranslational modifications of clock proteins modulate circadian rhythms and are thought to convert physiological signals into changes in protein regulatory function. To explore reversible lysine acetylation that is dependent on the clock, we have characterized the circadian acetylome in WT and Clock-deficient (Clock(-/-)) mouse liver by quantitative mass spectrometry. Our analysis revealed that a number of mitochondrial proteins involved in metabolic pathways are heavily influenced by clock-driven acetylation. Pathways such as glycolysis/gluconeogenesis, citric acid cycle, amino acid metabolism, and fatty acid metabolism were found to be highly enriched hits. The significant number of metabolic pathways whose protein acetylation profile is altered in Clock(-/-) mice prompted us to link the acetylome to the circadian metabolome previously characterized in our laboratory. Changes in enzyme acetylation over the circadian cycle and the link to metabolite levels are discussed, revealing biological implications connecting the circadian clock to cellular metabolic state.

  15. Genes regulated in neurons undergoing transcription-dependent apoptosis belong to signaling pathways rather than the apoptotic machinery.

    PubMed

    Desagher, Solange; Severac, Dany; Lipkin, Alexey; Bernis, Cyril; Ritchie, William; Le Digarcher, Anne; Journot, Laurent

    2005-02-18

    Neuronal apoptosis has been shown to require de novo RNA/protein synthesis. However, very few genes whose expression is necessary for inducing apoptosis have been identified so far. To systematically identify such genes, we have used genome-scale, long oligonucleotide microarrays and characterized the gene expression profile of cerebellar granule neurons in the early phase of apoptosis elicited by KCl deprivation. We identified 368 significantly differentially expressed genes, including most of the genes previously reported to be transcriptionally regulated in this paradigm. In addition, we identified several hundreds of genes whose transcriptional regulation has never been associated with neuronal apoptosis. We used automated Gene Ontology annotation, analysis of promoter sequences, and statistical tools to characterize these regulations. Although differentially expressed genes included some components of the apoptotic machinery, this functional category was not significantly over-represented among regulated genes. On the other hand, categories related to signal transduction were the most significantly over-represented group. This indicates that the apoptotic machinery is mainly constitutive, whereas molecular pathways that lead to the activation of apoptotic components are transcriptionally regulated. In particular, we show for the first time that signaling pathways known to be involved in the control of neuronal survival are regulated at the transcriptional level and not only by post-translational mechanisms. Moreover, our approach provides insights into novel transcription factors and novel mechanisms, such as the unfolded protein response and cell adhesion, that may contribute to the induction of neuronal apoptosis.

  16. Deltamethrin-induced oxidative stress and mitochondrial caspase-dependent signaling pathways in murine splenocytes.

    PubMed

    Kumar, Anoop; Sasmal, D; Bhaskar, Amand; Mukhopadhyay, Kunal; Thakur, Aman; Sharma, Neelima

    2016-07-01

    Deltamethrin (DLM) is a well-known pyrethroid insecticide used extensively in pest control. Exposure to DLM has been demonstrated to cause apoptosis in various cells. However, the immunotoxic effects of DLM on mammalian system and its mechanism is still an open question to be explored. To explore these effects, this study has been designed to first observe the interactions of DLM to immune cell receptors and its effects on the immune system. The docking score revealed that DLM has strong binding affinity toward the CD45 and CD28 receptors. In vitro study revealed that DLM induces apoptosis in murine splenocytes in a concentration-dependent manner. The earliest markers of apoptosis such as enhanced reactive oxygen species and caspase 3 activation are evident as early as 1 h by 25 and 50 µM DLM. Western blot analysis demonstrated that p38 MAP kinase and Bax expression is increased in a concentration-dependent manner, whereas Bcl 2 expression is significantly reduced after 3 h of DLM treatment. Glutathione depletion has been also observed at 3 and 6 h by 25 and 50 µM concentration of DLM. Flow cytometry results imply that the fraction of hypodiploid cells has gradually increased with all the concentrations of DLM at 18 h. N-acetyl cysteine effectively reduces the percentage of apoptotic cells, which is increased by DLM. In contrast, buthionine sulfoxamine causes an elevation in the percentage of apoptotic cells. Phenotyping data imply the effect of DLM toxicity in murine splenocytes. In brief, the study demonstrates that DLM causes apoptosis through its interaction with CD45 and CD28 receptors, leading to oxidative stress and activation of the mitochondrial caspase-dependent pathways which ultimately affects the immune functions. This study provides mechanistic information by which DLM causes toxicity in murine splenocytes. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 808-819, 2016.

  17. Topical Administration of the Mitochondrial PTP Opening Inhibitor CoQ10 Prevents Apoptotic Cell Death Induced by UVC-Irradiation in Rat's Corneas and Rabbit's Retinas

    NASA Astrophysics Data System (ADS)

    Papucci, Laura; Witort, Ewa; Schiavone, Nicola; Donnini, Martino; Lapucci, Andrea; Lulli, Matteo; Lazzarano, Stefano; Simoncini, Madine; Mazzoni, Tiziano; Falciani, Piergiuseppe; Capaccioli, Sergio

    2008-06-01

    We have previously demonstrated in vitro that ubiquitous free radical scavenger coenzyme Q10 prevents keratocyte apoptosis induced by excimer laser irradiation more efficiently than other antioxidants. We showed that its anti apoptotic property is independent of its free radical scavenging ability and is related to direct inhibition of PTP (permeability transition pore) opening and mitochondrial depolarization. Here, we demonstrate in vivo, that CoQ10 can efficiently protect rat's corneas and rabbit's retinas irradiated with UVC. Therefore, we propose application of CoQ10 as countermeasure to prevent micro radiation-induced eye damage during interplanetary space exploration.

  18. Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway

    SciTech Connect

    Chen, T.-G.; Chen, T.-L.; Chang, H.-C.; Tai, Y.-T.; Cherng, Y.-G.; Chang, Y.-T.; Chen, R.-M. . E-mail: rmchen@tmu.edu.tw

    2007-02-15

    Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, ox

  19. Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway.

    PubMed

    Chen, Tyng-Guey; Chen, Ta-Liang; Chang, Huai-Chia; Tai, Yu-Ting; Cherng, Yih-Giun; Chang, Ya-Ting; Chen, Ruei-Ming

    2007-02-15

    Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, ox

  20. Diallyl trisulfide induces apoptosis of human basal cell carcinoma cells via endoplasmic reticulum stress and the mitochondrial pathway.

    PubMed

    Wang, Hsiao-Chi; Hsieh, Shu-Chen; Yang, Jen-Hung; Lin, Shuw-Yuan; Sheen, Lee-Yan

    2012-01-01

    Diallyl trisulfide (DATS), an active component of garlic oil, has attracted much attention because of its anticancer effect on several types of cancers. However, the mechanism of DATS-induced apoptosis of basal cell carcinoma (BCC) is not fully understood. In the present study, we revealed that DATS-mediated dose-dependent induction of apoptosis in BCC cells was associated with intracellular reactive oxygen species accumulation and disrupted mitochondrial membrane potential. Western analysis demonstrated concordant expression of molecules involved in mitochondrial apoptosis, including DATS-associated increases in phospho-p53, proapoptotic Bax, and decreases in antiapoptotic Bcl-2 and Bcl-xl in BCC cells. Moreover, DATS induced the release of cytochrome c, apoptosis-inducing factor, and HtrA2/Omi into the cytoplasm, and activated factors downstream of caspase-dependent and caspase-independent apoptosis, including nuclear translocation of apoptotic-inducing factor and endonuclease G and the caspase cascade. These results were confirmed by pretreatment with the antioxidant N-acetyl-L-cysteine and the caspase inhibitor (z-VAD-fmk), the latter of which did not completely enhance the viability of DATS-treated BBC cells. Exposure to DATS additionally induced endogenous endoplasmic reticulum stress markers and intracellular Ca2⁺ mobilization, upregulation of Bip/GRP78 and CHOP/GADD153, and activation of caspase-4. Our findings suggest that DATS exerts chemopreventive potential via ER stress and the mitochondrial pathway in BCC cells.

  1. In vitro cytotoxicity of Mokko lactone in human leukemia HL-60 cells: induction of apoptotic cell death by mitochondrial membrane potential collapse.

    PubMed

    Yun, Y G; Oh, H; Oh, G S; Pae, H O; Choi, B M; Kwon, J W; Kwon, T O; Jang, S I; Chung, Hun-Taeg

    2004-08-01

    We studied the effect of mokko lactone (ML) isolated from the roots of Saussurea lappa (Compositae), a plant that is used for medicinal purposes in Korea, on the induction of apoptosis in human leukemia HL-60 cells. ML was cytotoxic to HL-60 cells, and this cytotoxic effect of ML appears to be attributable to its induction of apoptotic cell death, as ML induced nuclear morphologic changes and internucleosomal DNA fragmentation and increased the proportion of Annexin V-positive cells and the activity of caspase-3. Further studies revealed that the induction of apoptosis by ML was associated with the loss of mitochondrial membrane potential. Collectively, our results suggest that apoptosis induced by ML in HL-60 cells was executed by a collapse of mitochondrial membrane potential followed by the activation of caspase-3. This is the first report on the mechanism of apoptosis-inducing effect of ML.

  2. Fucoidan derived from Undaria pinnatifida induces apoptosis in human hepatocellular carcinoma SMMC-7721 cells via the ROS-mediated mitochondrial pathway.

    PubMed

    Yang, Lili; Wang, Peisheng; Wang, Huaxin; Li, Qiaomei; Teng, Hongming; Liu, Zhichao; Yang, Wenbo; Hou, Lin; Zou, Xiangyang

    2013-06-10

    Fucoidans, fucose-enriched sulfated polysaccharides isolated from brown algae and marine invertebrates, have been shown to exert anticancer activity in several types of human cancer, including leukemia and breast cancer and in lung adenocarcinoma cells. In the present study, the anticancer activity of the fucoidan extracted from the brown seaweed Undaria pinnatifida was investigated in human hepatocellular carcinoma SMMC-7721 cells, and the underlying mechanisms of action were investigated. SMMC-7721 cells exposed to fucoidan displayed growth inhibition and several typical features of apoptotic cells, such as chromatin condensation and marginalization, a decrease in the number of mitochondria, and in mitochondrial swelling and vacuolation. Fucoidan-induced cell death was associated with depletion of reduced glutathione (GSH), accumulation of high intracellular levels of reactive oxygen species (ROS), and accompanied by damage to the mitochondrial ultrastructure, depolarization of the mitochondrial membrane potential (MMP, Δψm) and caspase activation. Moreover, fucoidan led to altered expression of factors related to apoptosis, including downregulating Livin and XIAP mRNA, which are members of the inhibitor of apoptotic protein (IAP) family, and increased the Bax-to-Bcl-2 ratio. These findings suggest that fucoidan isolated from U. pinnatifida induced apoptosis in SMMC-7721 cells via the ROS-mediated mitochondrial pathway.

  3. The H63D HFE gene variant promotes activation of the intrinsic apoptotic pathway via mitochondria dysfunction following β-amyloid peptide exposure.

    PubMed

    Mairuae, Nootchanat; Hall Ii, Eric C; Cheepsunthorn, Poonlarp; Lee, Sang Y; Connor, James R

    2010-11-01

    Numerous epidemiological studies suggest that the expression of the HFE allelic variant H63D may be a risk factor or genetic modifier for Alzheimer's disease (AD). The H63D variant alters cellular iron homeostasis and increases baseline oxidative stress. The elevated cellular stress milieu, we have proposed, may alter cellular responses to genetic and environmental determinants of AD. Accumulation of β-amyloid peptides (Aβ) is one of the most prominent pathogenic characteristics of AD. Several studies have demonstrated that Aβ can induce neuronal cell death through apoptosis. In this study, we provide evidence that an Aβ(25-35) fragment, which contains the cytotoxic sequence of the amyloid peptide, activates the intrinsic apoptotic pathway in SH-SY5Y human neuroblastoma cells expressing the HFE allelic variant H63D to a greater extent than in cells with wild-type (WT) HFE. Specifically, Aβ(25-35) peptide exposure significantly induced Bax translocation from the cytosol to the mitochondria in H63D-expressing cells compared with WT cells. This translocation was associated with increased cytochrome c release from mitochondria and an increase in active caspase-9 and caspase-3 activity in H63D cells. Consequently, there is increased apoptosis in cells expressing the H63D variant as opposed to cells expressing WT HFE. We also found increased amyloid precursor protein (APP) and Aβ(1-42) peptide in the mitochondrial compartment as well as increased mitochondrial stress in H63D-expressing cells compared with WT. These findings support our hypothesis that the presence of the HFE H63D allele enables factors that trigger neurodegenerative processes associated with AD and predisposes cells to cytotoxcity.

  4. ROS generation mediates the anti-cancer effects of WZ35 via activating JNK and ER stress apoptotic pathways in gastric cancer

    PubMed Central

    Zou, Peng; Zhang, Junru; Xia, Yiqun; Kanchana, Karvannan; Guo, Guilong; Chen, Wenbo; Huang, Yi; Wang, Zhe; Yang, Shulin; Liang, Guang

    2015-01-01

    Gastric cancer is one of the leading causes of cancer mortality in the world, and finding novel agents and strategies for the treatment of advanced gastric cancer is of urgent need. Curcumin is a well-known natural product with anti-cancer ability, but is limited by its poor chemical stability. In this study, an analog of curcumin with high chemical stability, WZ35, was designed and evaluated for its anti-cancer effects and underlying mechanisms against human gastric cancer. WZ35 showed much stronger anti-proliferative effects than curcumin, accompanied by dose-dependent induction of cell cycle arrest and apoptosis in gastric cancer cells. Mechanistically, our data showed that WZ35 induced reactive oxygen species (ROS) production, resulting in the activation of both JNK-mitochondrial and ER stress apoptotic pathways and eventually cell apoptosis in SGC-7901 cells. Blockage of ROS production totally reversed WZ35-induced JNK and ER stress activation as well as cancer cell apoptosis. In vivo, WZ35 showed a significant reduction in SGC-7901 xenograft tumor size in a dose-dependent manner. Taken together, this work provides a novel anticancer candidate for the treatment of gastric cancer, and importantly, reveals that increased ROS generation might be an effective strategy in human gastric cancer treatment. PMID:25714022

  5. Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways

    PubMed Central

    Chen, Li-Gang; Liu, Yuan-Sheng; Zheng, Tang-Hui; Chen, Xu; Li, Ping; Xiao, Chuan-Xing; Ren, Jian-Lin

    2016-01-01

    A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro. PMID:27900002

  6. Nesfatin-1 protects dopaminergic neurons against MPP+/MPTP-induced neurotoxicity through the C-Raf–ERK1/2-dependent anti-apoptotic pathway

    PubMed Central

    Shen, Xiao-Li; Song, Ning; Du, Xi-Xun; Li, Yong; Xie, Jun-Xia; Jiang, Hong

    2017-01-01

    Several brain-gut peptides have been reported to have a close relationship with the central dopaminergic system; one such brain-gut peptide is nesfatin-1. Nesfatin-1 is a satiety peptide that is predominantly secreted by X/A-like endocrine cells in the gastric glands, where ghrelin is also secreted. We previously reported that ghrelin exerted neuroprotective effects on nigral dopaminergic neurons, which implied a role for ghrelin in Parkinson’s disease (PD). In the present study, we aim to clarify whether nesfatin-1 has similar effects on dopaminergic neurons both in vivo and in vitro. We show that nesfatin-1 attenuates the loss of nigral dopaminergic neurons in the 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. In addition, nesfatin-1 antagonized 1-methyl-4-phenylpyridillium ion (MPP+)-induced toxicity by restoring mitochondrial function, inhibiting cytochrome C release and preventing caspase-3 activation in MPP+-treated MES23.5 dopaminergic cells. These neuroprotective effects could be abolished by selective inhibition of C-Raf and the extracellular signal-regulated protein kinase 1/2 (ERK1/2). Our data suggest that C-Raf-ERK1/2, which is involved in an anti-apoptotic pathway, is responsible for the neuroprotective effects of nesfatin-1 in the context of MPTP-induced toxicity. These results imply that nesfatin-1 might have therapeutic potential for PD. PMID:28106099

  7. Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways.

    PubMed

    Chen, Li-Gang; Liu, Yuan-Sheng; Zheng, Tang-Hui; Chen, Xu; Li, Ping; Xiao, Chuan-Xing; Ren, Jian-Lin

    2016-11-01

    A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro.

  8. Cinnamaldehyde-induced apoptosis in human hepatoma PLC/PRF/5 cells involves the mitochondrial death pathway and is sensitive to inhibition by cyclosporin A and z-VAD-fmk.

    PubMed

    Lin, Liang-Tzung; Tai, Chen-Jei; Chang, Shun-Pang; Chen, Jin-Liang; Wu, Shu-Jing; Lin, Chun-Ching

    2013-12-01

    Cinnamaldehyde (CIN) has been shown to exert chemopreventive activity against several types of human cancer cells. We previously reported that CIN induced apoptosis of human hepatoma PLC/PRF/5 cells and this effect was associated with activation of the pro-apoptotic Bcl-2 family of proteins and the MAPK cascade. To further clarify the underlying mechanism of CIN-induced apoptosis, we examined in this study its relationship with the mitochondrial death pathway using the mitochondrial permeability transition (MPT) inhibitor, cyclosporin A (CsA), and the general caspase inhibitor, z-VAD-fmk. Results indicated that CIN-induced apoptosis involved enhanced ROS generation, disruption of mitochondrial potential, and the mitochondrial release of cytochrome c and Smac/DIABLO into the cytosol, which in turn promoted caspase-3 to its active form and the subsequent cleavage of PARP. Treatment with CIN also downregulated protein levels of the anti-apoptotic factors XIAP and Bcl-2 with concomitant accumulation of the pro-apoptotic Bax in a timedependent manner. These mitochondria-related apoptotic effects induced by CIN were however blocked by CsA and z-VAD-fmk pretreatments, which prevented cells from undergoing programmed cell death triggered by CIN. Furthermore, the increase of Bax and decrease of Bcl-2 and XIAP protein expression due to CIN treatment were also reversely modulated by the two inhibitors. Taken together, these results suggested that CIN is an apoptotic inducer that acts on the mitochondrial death pathway in PLC/PRF/5 cells and its effect could be blocked by CsA and z-VAD-fmk.

  9. Involvement of caspase-12-dependent apoptotic pathway in ionic radiocontrast urografin-induced renal tubular cell injury

    SciTech Connect

    Wu, Cheng Tien; Weng, Te I.; Chen, Li Ping; Chiang, Chih Kang; Liu, Shing Hwa

    2013-01-01

    Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect subjects in the disorder of CM-induced nephropathy. Our previous work has demonstrated that CM shows to activate the endoplasmic reticulum (ER)-related adaptive unfolding protein response (UPR) activators. Glucose-regulated protein 78 (GRP78)/eukaryotic initiation factor 2α (eIF2α)-related pathways play a protective role during the urografin (an ionic CM)-induced renal tubular injury. However, the involvement of ER stress-related apoptotic signals in the urografin-induced renal tubular cell injury remains unclear. Here, we examined by the in vivo and in vitro experiments to explore whether ER stress-regulated pro-apoptotic activators participate in urografin-induced renal injury. Urografin induced renal tubular dilation, tubular cells detachment, and necrosis in the kidneys of rats. The tubular apoptosis, ER stress-related pro-apoptotic transcriptional factors, and kidney injury marker-1 (kim-1) were also conspicuously up-regulated in urografin-treated rats. Furthermore, treatment of normal rat kidney (NRK)-52E tubular cells with urografin augmented the expressions of activating transcription factor-6 (ATF-6), C/EBP homologous protein (CHOP), Bax, caspase-12, JNK, and inositol-requiring enzyme (IRE) 1 signals. Urografin-induced renal tubular cell apoptosis was not reversed by the inhibitors of ATF-6, JNK signals or CHOP siRNA transfection, but it could be partially reversed by the inhibitor of caspase-12. Taken together, the present results and our previous findings suggest that exposure of CM/urografin activates the ER stress-regulated survival- and apoptosis-related signaling pathways in renal tubular cells. Caspase-12-dependent apoptotic pathway may be partially involved in the urografin-induced nephropathy. -- Highlights: ► Ionic contrast medium-urografin induces renal tubular cell apoptosis. ► Urografin induces the ER stress-regulated survival and apoptosis

  10. Interferon α Induces the Apoptosis of Cervical Cancer HeLa Cells by Activating both the Intrinsic Mitochondrial Pathway and Endoplasmic Reticulum Stress-Induced Pathway

    PubMed Central

    Shi, Wei-Ye; Cao, Cheng; Liu, Li

    2016-01-01

    The interferon α (IFN-α) has been often used as a sensitizing agent for the treatment of various malignancies such as hepatocellular carcinoma, malignant melanoma, and renal cell cancer by promoting the apoptosis of thesetumor cell types. However, the effect of IFN-α on cervical cancer remains unknown. In this study, HeLa cells were used as a testing model for the treatment of IFN-α on cervical cancer. The results indicate that IFN-α markedly inhibits the proliferation and induces the apoptosis of HeLa cells. The activation of caspase 3, the up-regulation of both Bim and cleaved poly (ADP-ribose) polymerase (PARP) 1, the down-regulation of Bcl-xL, as well as the release of cytochrome c from mitochondria were significantly induced upon IFN-α treatment, indicating that the intrinsic apoptotic pathway could be activated by IFN-α treatment. In addition, caspase 4—which is involved in the endoplasmic reticulum (ER) stress-induced apoptosis—was activated in response to IFN-α treatment. Knocking down caspase 4 by small interfering RNA (siRNA) markedly reduced the IFN-α-mediated cell apoptosis. However, no significant changes in the expressions of caspases 8 and 10 were observed upon IFN-α treatment, indicating that the apoptosis caused by IFN-α might be independent of the extrinsic apoptotic pathway. These findings suggest that IFN-α may possess anti-cervical cancer capacity by activating cell apoptosis via the intrinsic mitochondrial pathway and caspase-4-related ER stress-induced pathway. PMID:27827850

  11. MicroRNA-125a promotes resistance to BRAF inhibitors through suppression of the intrinsic apoptotic pathway.

    PubMed

    Koetz-Ploch, Lisa; Hanniford, Douglas; Dolgalev, Igor; Sokolova, Elena; Zhong, Judy; Díaz-Martínez, Marta; Bernstein, Emily; Darvishian, Farbod; Flaherty, Keith T; Chapman, Paul B; Tawbi, Hussein; Hernando, Eva

    2017-01-31

    Melanoma patients with BRAF(V)(600E) -mutant tumors display striking responses to BRAF inhibitors (BRAFi); however, almost all invariably relapse with drug-resistant disease. Here we report that microRNA-125a (miR-125a) expression is upregulated in human melanoma cells and patient tissues upon acquisition of BRAFi resistance. We show that miR-125a induction confers resistance to BRAF(V)(600E) melanoma cells to BRAFi by directly suppressing pro-apoptotic components of the intrinsic apoptosis pathway, including BAK1 and MLK3. Apoptotic suppression and prolonged survival favor reactivation of the MAPK and AKT pathways by drug-resistant melanoma cells. We demonstrate that miR-125a inhibition suppresses the emergence of resistance to BRAFi and, in a subset of resistant melanoma cell lines, leads to partial drug re-sensitization. Finally, we show that miR-125a upregulation is mediated by TGFβ signaling. In conclusion, the identification of this novel role for miR-125a in BRAFi resistance exposes clinically relevant mechanisms of melanoma cell survival that can be exploited therapeutically. This article is protected by copyright. All rights reserved.

  12. Mitochondrial Gene Expression Profiles and Metabolic Pathways in the Amygdala Associated with Exaggerated Fear in an Animal Model of PTSD

    PubMed Central

    Li, He; Li, Xin; Smerin, Stanley E.; Zhang, Lei; Jia, Min; Xing, Guoqiang; Su, Yan A.; Wen, Jillian; Benedek, David; Ursano, Robert

    2014-01-01

    The metabolic mechanisms underlying the development of exaggerated fear in post-traumatic stress disorder (PTSD) are not well defined. In the present study, alteration in the expression of genes associated with mitochondrial function in the amygdala of an animal model of PTSD was determined. Amygdala tissue samples were excised from 10 non-stressed control rats and 10 stressed rats, 14 days post-stress treatment. Total RNA was isolated, cDNA was synthesized, and gene expression levels were determined using a cDNA microarray. During the development of the exaggerated fear associated with PTSD, 48 genes were found to be significantly upregulated and 37 were significantly downregulated in the amygdala complex based on stringent criteria (p < 0.01). Ingenuity pathway analysis revealed up- or downregulation in the amygdala complex of four signaling networks – one associated with inflammatory and apoptotic pathways, one with immune mediators and metabolism, one with transcriptional factors, and one with chromatin remodeling. Thus, informatics of a neuronal gene array allowed us to determine the expression profile of mitochondrial genes in the amygdala complex of an animal model of PTSD. The result is a further understanding of the metabolic and neuronal signaling mechanisms associated with delayed and exaggerated fear. PMID:25295026

  13. Synergistic effect of fisetin combined with sorafenib in human cervical cancer HeLa cells through activation of death receptor-5 mediated caspase-8/caspase-3 and the mitochondria-dependent apoptotic pathway.

    PubMed

    Lin, Ming-Te; Lin, Chia-Liang; Lin, Tzu-Yu; Cheng, Chun-Wen; Yang, Shun-Fa; Lin, Chu-Liang; Wu, Chih-Chien; Hsieh, Yi-Hsien; Tsai, Jen-Pi

    2016-05-01

    Combining antitumor agents with bioactive compounds is a potential strategy for improving the effect of chemotherapy on cancer cells. The goal of this study was to elucidate the antitumor effect of the flavonoid, fisetin, combined with the multikinase inhibitor, sorafenib, against human cervical cancer cells in vitro and in vivo. The combination of fisetin and sorafenib synergistically induced apoptosis in HeLa cells, which is accompanied by a marked increase in loss of mitochondrial membrane potential. Apoptosis induction was achieved by caspase-3 and caspase-8 activation which increased the ratio of Bax/Bcl-2 and caused the subsequent cleavage of PARP level while disrupting the mitochondrial membrane potential in HeLa cells. Decreased Bax/Bcl-2 ratio level and mitochondrial membrane potential were also observed in siDR5-treated HeLa cells. In addition, in vivo studies revealed that the combined fisetin and sorafenib treatment was clearly superior to sorafenib treatment alone using a HeLa xenograft model. Our study showed that the combination of fisetin and sorafenib exerted better synergistic effects in vitro and in vivo than either agent used alone against human cervical cancer, and this synergism was based on apoptotic potential through a mitochondrial- and DR5-dependent caspase-8/caspase-3 signaling pathway. This combined fisetin and sorafenib treatment represents a novel therapeutic strategy for further clinical developments in advanced cervical cancer.

  14. The Nrf2/ARE Pathway: A Promising Target to Counteract Mitochondrial Dysfunction in Parkinson's Disease

    PubMed Central

    Tufekci, Kemal Ugur; Civi Bayin, Ezgi; Genc, Sermin; Genc, Kursad

    2011-01-01

    Mitochondrial dysfunction is a prominent feature of various neurodegenerative diseases as strict regulation of integrated mitochondrial functions is essential for neuronal signaling, plasticity, and transmitter release. Many lines of evidence suggest that mitochondrial dysfunction plays a central role in the pathogenesis of Parkinson's disease (PD). Several PD-associated genes interface with mitochondrial dynamics regulating the structure and function of the mitochondrial network. Mitochondrial dysfunction can induce neuron death through a plethora of mechanisms. Both mitochondrial dysfunction and neuroinflammation, a common denominator of PD, lead to an increased production of reactive oxygen species, which are detrimental to neurons. The transcription factor nuclear factor E2-related factor 2 (Nrf2, NFE2L2) is an emerging target to counteract mitochondrial dysfunction and its consequences in PD. Nrf2 activates the antioxidant response element (ARE) pathway, including a battery of cytoprotective genes such as antioxidants and anti-inflammatory genes and several transcription factors involved in mitochondrial biogenesis. Here, the current knowledge about the role of mitochondrial dysfunction in PD, Nrf2/ARE stress-response mechanisms, and the evidence for specific links between this pathway and PD are summarized. The neuroprotection of nigral dopaminergic neurons by the activation of Nrf2 through several inducers in PD is also emphasized as a promising therapeutic approach. PMID:21403858

  15. The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process

    SciTech Connect

    Park, Jeehye; Lee, Gina; Chung, Jongkyeong

    2009-01-16

    The two Parkinson's disease (PD) genes, PTEN-induced kinase 1 (PINK1) and parkin, are linked in a common pathway which affects mitochondrial integrity and function. However, it is still not known what this pathway does in the mitochondria. Therefore, we investigated its physiological function in Drosophila. Because Drosophila PINK1 and parkin mutants show changes in mitochondrial morphology in both indirect flight muscles and dopaminergic neurons, we here investigated whether the PINK1-Parkin pathway genetically interacts with the regulators of mitochondrial fusion and fission such as Drp1, which promotes mitochondrial fission, and Opa1 or Marf, which induces mitochondrial fusion. Surprisingly, DrosophilaPINK1 and parkin mutant phenotypes were markedly suppressed by overexpression of Drp1 or downregulation of Opa1 or Marf, indicating that the PINK1-Parkin pathway regulates mitochondrial remodeling process in the direction of promoting mitochondrial fission. Therefore, we strongly suggest that mitochondrial fusion and fission process could be a prominent therapeutic target for the treatment of PD.

  16. Upstream Pathways Controlling Mitochondrial Function in Major Psychosis

    PubMed Central

    Machado, Alencar Kolinski; Pan, Alexander Yongshuai; da Silva, Tatiane Morgana; Duong, Angela

    2016-01-01

    Mitochondrial dysfunction is commonly observed in bipolar disorder (BD) and schizophrenia (SCZ) and may be a central feature of psychosis. These illnesses are complex and heterogeneous, which is reflected by the complexity of the processes regulating mitochondrial function. Mitochondria are typically associated with energy production; however, dysfunction of mitochondria affects not only energy production but also vital cellular processes, including the formation of reactive oxygen species, cell cycle and survival, intracellular Ca2+ homeostasis, and neurotransmission. In this review, we characterize the upstream components controlling mitochondrial function, including 1) mutations in nuclear and mitochondrial DNA, 2) mitochondrial dynamics, and 3) intracellular Ca2+ homeostasis. Characterizing and understanding the upstream factors that regulate mitochondrial function is essential to understand progression of these illnesses and develop biomarkers and therapeutics. PMID:27310240

  17. Withania somnifera alleviates parkinsonian phenotypes by inhibiting apoptotic pathways in dopaminergic neurons.

    PubMed

    Prakash, Jay; Chouhan, Shikha; Yadav, Satyndra Kumar; Westfall, Susan; Rai, Sachchida Nand; Singh, Surya Pratap

    2014-12-01

    Maneb (MB) and paraquat (PQ) are environmental toxins that have been experimentally used to induce selective damage of dopaminergic neurons leading to the development of Parkinson's disease (PD). Although the mechanism of this selective neuronal toxicity in not fully understood, oxidative stress has been linked to the pathogenesis of PD. The present study investigates the mechanisms of neuroprotection elicited by Withania somnifera (Ws), a herb traditionally recognized by the Indian system of medicine, Ayurveda. An ethanolic root extract of Ws was co-treated with the MB-PQ induced mouse model of PD and was shown to significantly rescue canonical indicators of PD including compromised locomotor activity, reduced dopamine in the substantia nigra and various aspects of oxidative damage. In particular, Ws reduced the expression of iNOS, a measure of oxidative stress. Ws also significantly improved the MB + PQ mediated induction of a pro-apoptotic state by reducing Bax and inducing Bcl-2 protein expression, respectively. Finally, Ws reduced expression of the pro-inflammatory marker of astrocyte activation, GFAP. Altogether, the present study suggests that Ws treatment provides nigrostriatal dopaminergic neuroprotection against MB-PQ induced Parkinsonism by the modulation of oxidative stress and apoptotic machinery possibly accounting for the behavioural effects.

  18. Downregulation of PI3-K/Akt/PTEN pathway and activation of mitochondrial intrinsic apoptosis by Diclofenac and Curcumin in colon cancer.

    PubMed

    Rana, Chandan; Piplani, Honit; Vaish, Vivek; Nehru, Bimla; Sanyal, S N

    2015-04-01

    Phosphatidylinositol 3-kinase (PI3-K)/PTEN/Akt signaling is over activated in various tumors including colon cancer. Activation of this pathway regulates multiple biological processes such as apoptosis, metabolism, cell proliferation, and cell growth that underlie the biology of a cancer cell. In the present study, the chemopreventive effects have been observed of Diclofenac, a preferential COX-2 inhibitory non-steroidal anti-inflammatory drugs, and Curcumin, a natural anti-inflammatory agent, in the early stage of colorectal carcinogenesis induced by 1,2-dimethylhydrazine dihydrochloride in rats. The tumor-promoting role of PI3-K/Akt/PTEN signal transduction pathway and its association with anti-apoptotic family of proteins are also observed. Both Diclofenac and Curcumin downregulated the PI3-K and Akt expression while promoting the apoptotic mechanism. Diclofenac and Curcumin administration significantly increased the expression of pro-apoptotic Bcl-2 family members (Bad and Bax) while decreasing the anti-apoptotic Bcl-2 protein. An up-regulation of cysteine protease family apoptosis executioner, such as caspase-3 and -9, is seen. Diclofenac and Curcumin inhibited the Bcl-2 protein by directly interacting at the active site by multiple hydrogen bonding, as also evident by negative glide score of Bcl-2. These drugs stimulated apoptosis by increasing reactive oxygen species (ROS) generation and simultaneously decreasing the mitochondrial membrane potential (ΔΨ M). Diclofenac and Curcumin showed anti-neoplastic effects by downregulating PI3-K/Akt/PTEN pathway, inducing apoptosis, increasing ROS generation, and decreasing ΔΨ M. The anti-neoplastic and apoptotic effects were found enhanced when both Diclofenac and Curcumin were administered together, rather than individually.

  19. Highly efficient synthetic iron-dependent nucleases activate both intrinsic and extrinsic apoptotic death pathways in leukemia cancer cells.

    PubMed

    Horn, Adolfo; Fernandes, Christiane; Parrilha, Gabrieli L; Kanashiro, Milton M; Borges, Franz V; de Melo, Edésio J T; Schenk, Gerhard; Terenzi, Hernán; Pich, Claus T

    2013-11-01

    The nuclease activity and the cytotoxicity toward human leukemia cancer cells of iron complexes, [Fe(HPClNOL)Cl2]NO3 (1), [Cl(HPClNOL)Fe(μ-O)Fe(HPClNOL)Cl]Cl2·2H2O (2), and [(SO4)(HPClNOL)Fe(μ-O)Fe(HPClNOL)(SO4)]·6H2O (3) (HPClNOL=1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol), were investigated. Each complex was able to promote plasmid DNA cleavage and change the supercoiled form of the plasmid to circular and linear ones. Kinetic data revealed that (1), (2) and (3) increase the rate of DNA hydrolysis about 278, 192 and 339 million-fold, respectively. The activity of the complexes was inhibited by distamycin, indicating that they interact with the minor groove of the DNA. The cytotoxic activity of the complexes toward U937, HL-60, Jukart and THP-1 leukemia cancer cells was studied employing 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), fluorescence and electronic transmission microscopies, flow cytometry and a cytochrome C release assay. Compound (2) has the highest activity toward cancer cells and is the least toxic for normal ones (i.e. peripheral blood mononuclear cells (PBMCs)). In contrast, compound (1) is the least active toward cancer cells but displays the highest toxicity toward normal cells. Transmission electronic microscopy indicates that cell death shows features typical of apoptotic cells, which was confirmed using the annexin V-FITC/PI (fluorescein isothiocyanate/propidium iodide) assay. Furthermore, our data demonstrate that at an early stage during the treatment with complex (2) mitochondria lose their transmembrane potential, resulting in cytochrome C release. A quantification of caspases 3, 9 (intrinsic apoptosis pathway) and caspase 8 (extrinsic apoptosis pathway) indicated that both the intrinsic (via mitochondria) and extrinsic (via death receptors) pathways are involved in the apoptotic stimuli.

  20. Myristicin from nutmeg induces apoptosis via the mitochondrial pathway and down regulates genes of the DNA damage response pathways in human leukaemia K562 cells.

    PubMed

    Martins, Célia; Doran, Carolina; Silva, Inês C; Miranda, Claudia; Rueff, José; Rodrigues, António S

    2014-07-25

    Myristicin, an allylbenzene, is a major active component of various spices, such as nutmeg and cinnamon, plants from the Umbelliferae family or in some essential oils, such as oils of clove or marjoram. Human exposure to myristicin is low but widespread due to consumption of these spices and essential oils, added to food (e.g. cola drinks) or in traditional medicine. Occasionally high dose exposure occurs, leading to various clinical symptoms, however the molecular mechanisms underlying them are unknown. Our previous studies revealed that myristicin is not genotoxic and yet presented apoptotic activity. Therefore, in this work we assessed the apoptotic mechanisms induced by myristicin in human leukaemia cells. In order to gain further insight on the potential of myristicin to modulate gene expression we also analysed alterations in expression of 84 genes associated with the DNA damage response pathway. The results obtained show that myristicin can induce apoptosis as characterised by alterations in the mitochondrial membrane potential, cytochrome c release, caspase-3 activation, PARP-cleavage and DNA fragmentation. The gene expression profile revealed an overall down regulation of DNA damage response genes after exposure to myristicin, with significant under-expression of genes associated with nucleotide excision repair (ERCC1), double strand break repair (RAD50, RAD51) and DNA damage signalling (ATM) and stress response (GADD45A, GADD45G). On the whole, we demonstrate that myristicin can alter mitochondrial membrane function, induce apoptosis and modulate gene expression in human leukaemia K562 cells. This study provides further detail on the molecular mechanisms underlying the biological activity of myristicin.

  1. Hydrogen peroxide induces apoptosis via a mitochondrial pathway in chondrocytes

    NASA Astrophysics Data System (ADS)

    Zhuang, Cai-ping; Liang, Qian; Wang, Xiao-ping; Chen, Tong-sheng

    2012-03-01

    The degenerative joint disease such as osteoarthritis (OA) is closely associated with the death of chondrocytes in apoptosis fashion. Hydrogen peroxide (H2O2), higher expression following acute damage in OA patients, has been shown to be up-regulated during apoptosis in a bulk of experimental models. This study was aimed to explore the mechanism of H2O2-induced rabbit chondrocytes apoptosis. Articular cartilage was biopsied from the joints of 6 weeks old New Zealand rabbits. Cell Counting Kit (CCK-8) assay was used to assess the inhibitory effect of H2O2 on cell viability. H2O2 treatment induced a remarkable reduction of cell viability. We used flow cytometry to assess the form of cell death with Annexin-V/PI double staining, and found that H2O2 treatment induced apoptosis in a dose-and time-dependent manner. Exposure of chondrocytes to 1.5 mM of H2O2 for 2 h induced a burst apoptosis that can be alleviated by N-acetyl cysteine (NAC) pretreatment, an anti-oxidant amino-acid derivative. Loss of mitochondria membrane potential (▵Ψm) was evaluated using confocal microscopy imaging and flow cytometry (FCM). H2O2 treatment induced a marked reduction of ▵Ψm, and the abrupt disappearance of ▵Ψm occurred within 5 minutes. These results indicate that H2O2 induces a rapid apoptosis via a mitochondrial pathway in rabbit chondrocytes.

  2. Bioactive natural products against prostate cancer: mechanism of action and autophagic/apoptotic molecular pathways.

    PubMed

    Gioti, Katerina; Tenta, Roxane

    2015-05-01

    Prostate cancer is one of the leading causes of death worldwide for men. There is increasing evidence that diet and lifestyle play a crucial role in prostate cancer biology and tumorigenesis. Due to the fact that conventional chemotherapy is not adequately effective against prostate cancer and has severe side effects, numerous in vitro studies have been conducted in order to identify the potent cytotoxic or chemopreventive activity of naturally occurring compounds and their respective molecular mechanisms of action. In this context, many natural compounds isolated from plants have been found to inhibit cancer growth and to induce cell cycle arrest, suppress angiogenesis, and promote apoptotic or autophagic cell death. Therefore, in this article, the most promising bioactive natural products and their respective mechanisms of action for the prevention or/and treatment of prostate cancer are presented.

  3. Regulation of apoptotic pathways by Stylophora pistillata (Anthozoa, Pocilloporidae) to survive thermal stress and bleaching.

    PubMed

    Kvitt, Hagit; Rosenfeld, Hanna; Zandbank, Keren; Tchernov, Dan

    2011-01-01

    Elevated seawater temperatures are associated with coral bleaching events and related mortality. Nevertheless, some coral species are able to survive bleaching and recover. The apoptotic responses associated to this ability were studied over 3 years in the coral Stylophora pistillata from the Gulf of Eilat subjected to long term thermal stress. These include caspase activity and the expression profiles of the S. pistillata caspase and Bcl-2 genes (StyCasp and StyBcl-2-like) cloned in this study. In corals exposed to thermal stress (32 or 34°C), caspase activity and the expression levels of the StyBcl-2-like gene increased over time (6-48 h) and declined to basal levels within 72 h of thermal stress. Distinct transcript levels were obtained for the StyCasp gene, with stimulated expression from 6 to 48 h of 34°C thermal stress, coinciding with the onset of bleaching. Increased cell death was detected in situ only between 6 to 48 h of stress and was limited to the gastroderm. The bleached corals survived up to one month at 32°C, and recovered back symbionts when placed at 24°C. These results point to a two-stage response in corals that withstand thermal stress: (i) the onset of apoptosis, accompanied by rapid activation of anti-oxidant/anti-apoptotic mediators that block the progression of apoptosis to other cells and (ii) acclimatization of the coral to the chronic thermal stress alongside the completion of symbiosis breakdown. Accordingly, the coral's ability to rapidly curb apoptosis appears to be the most important trait affecting the coral's thermotolerance and survival.

  4. E-Cigarette Vapor Induces an Apoptotic Response in Human Gingival Epithelial Cells Through the Caspase-3 Pathway.

    PubMed

    Rouabhia, Mahmoud; Park, Hyun Jin; Semlali, Abdelhabib; Zakrzewski, Andrew; Chmielewski, Witold; Chakir, Jamila

    2017-06-01

    Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E-cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e-cigarette vapor on human gingival epithelial cells. Results show that e-cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e-cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E-cigarette vapor also increased L-lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e-cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e-cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase-3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase-3 inhibitor. The adverse effects of e-cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. J. Cell. Physiol. 232: 1539-1547, 2017. © 2016 Wiley Periodicals, Inc.

  5. Inhibition of anti-apoptotic signals by Wortmannin induces apoptosis in the remote myocardium after LAD ligation: evidence for a protein kinase C-δ-dependent pathway.

    PubMed

    Wiedemann, Stephan; Wessela, Teresa; Schwarz, Kerstin; Joachim, Dirk; Jercke, Marcel; Strasser, Ruth H; Ebner, Bernd; Simonis, Gregor

    2013-01-01

    It has been shown that, in the remote myocardium after infarction (MI), protein kinase C (PKC) inhibition reduces apoptosis both by blocking proapoptotic pathways and by activating antiapoptotic signals including the Akt pathway. However, it was open if vice versa, blockade of antiapoptotic pathways may influence proapoptotic signals. To clarify this, the present study tested the effects of the PI3-kinase blocker Wortmannin on proapoptotic signals and on apoptosis execution in the remote myocardium after infarction. Rats were subjected to MI by LAD ligation in situ. Some were pre-treated with Wortmannin alone or in combination with the PKC inhibitor Chelerythrine. After 24 h, pro- and anti-apoptotic signals (caspase-3, PKC isoforms, p38-MAPK, p42/44-MAPK, Akt, Bad), and marker of apoptosis execution (TUNEL) were quantified in the myocardium remote from the infarction. Wortmannin treatment increased apoptosis in the remote myocardium both at baseline and after MI, together with an activation of the PKC-δ/p38-MAPK-pathway. PKC-ε and p42/44-MAPK were unaffected. Combined treatment with Wortmannin and Chelerythrine fully reversed the pro-apoptotic effects of Wortmannin both at baseline and after MI. The PKC-δ-p38-MAPK-pathway as a strong signal for apoptosis in the non-infarcted myocardium can be influenced by targeting the anti-apoptotic PI3-kinase pathway. This gives evidence of a bi-directional crosstalk of pro- and anti-apoptotic signals after infarction.

  6. Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway.

    PubMed

    Lin, Meng-Liang; Lu, Yao-Cheng; Chung, Jing-Gung; Li, Yi-Chen; Wang, Shyang-Guang; N G, Sue-Hwee; Wu, Chia-Yin; Su, Hong-Lin; Chen, Shih-Shun

    2010-05-01

    Aloe-emodin (AE), a natural, biologically active compound from the rhizome of Rheum palmatum, has been shown to induce apoptosis in several cancer cell lines in vitro. However, its molecular mechanism of action in the apoptosis induction of human nasopharyngeal carcinoma (NPC) cells has not been explored. This study shows that AE induced G(2)/M phase arrest by increasing levels of cyclin B1 bound to Cdc2, and also caused an increase in apoptosis of NPC cells, which was characterized by morphological changes, nuclear condensation, DNA fragmentation, caspase-3 activation, cleavage of poly (ADP-ribose) polymerase (PARP) and increased sub-G(1) population. Treatment of NPC cells with AE also resulted in a decrease in Bcl-X(L) and an increase in Bax expression. Ectopic expression of Bcl-X(L) but not Bcl-2 or small interfering RNA (siRNA)-mediated attenuation of Bax suppressed AE-induced apoptotic cell death. AE-induced loss of mitochondrial membrane potential (MMP) and increase in cellular Ca(++) content, reactive oxygen species (ROS) and apoptotic cell death were suppressed by the treatment of cyclosporin A (CsA) or caspase-8 inhibitor Z-IETD-FMK. Co-treatment with caspase-9 inhibitor Z-LEHD-FMK could inhibit AE-induced cell death and the activation of caspase-3 and -9. In addition, suppression of caspase-8 with the specific inhibitor Z-IETD-FMK inhibited AE-induced the activation of Bax, the cleavage of Bid, the translocation of tBid to the mitochondria and the release of cytochrome c, apoptosis-inducing factor (AIF) and Endo G from the mitochondria and subsequent apoptosis. Taken together, these results indicate that the caspase-8-mediated activation of the mitochondrial death pathway plays a critical role in AE-induced apoptosis of NPC cells.

  7. Interconnections between apoptotic and autophagic pathways during thiopurine-induced toxicity in cancer cells: the role of reactive oxygen species

    PubMed Central

    Chaabane, Wiem; Appell, Malin Lindqvist

    2016-01-01

    Thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine) are a class of genotoxic drugs extensively used in the treatment of various illnesses including leukemia. Their underlying molecular mechanism of action involves the activation of apoptosis and autophagy but remains widely unclear. Here we present evidence that autophagy induction by thiopurines is a survival mechanism that antagonizes apoptosis and is involved in degrading damaged mitochondria through mitophagy. On the other hand, apoptosis is the main cell death mechanism by thiopurines as its inhibition prohibited cell death. Thus a tight interplay between apoptosis and autophagy controls cell fate in response to thiopurine treatment. Moreover, thiopurines disrupt mitochondrial function and induce a loss of the mitochondrial transmembrane potential. The involvement of the mitochondrial pathway in thiopurine-induced apoptosis was further confirmed by increased formation of reactive oxygen species (ROS). Inhibiting oxidative stress protected the cells from thiopurine-induced cell death and ROS scavenging prohibited autophagy induction by thiopurines. Our data indicate that the anticarcinogenic effects of thiopurines are mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy. PMID:27689330

  8. The mitochondrial and endoplasmic reticulum pathways involved in the apoptosis of bursa of Fabricius cells in broilers exposed to dietary aflatoxin B1

    PubMed Central

    Fang, Jing; Liang, Na; Zhou, Mingqiang; Huang, Cheng; Peng, Xi

    2016-01-01

    Aflatoxin B1 (AFB1), a toxic metabolite produced by some fungi, exerts well-known hepatocarcinogenic and immunosuppressive effects, the latter can increase the apoptotic immune cells in vitro. However, it is largely unknown that which signaling pathways contribute to excessive apoptosis of immune cells which induced by AFB1. In this study, we investigated the roles of the mitochondria, endoplasmic reticulum (ER) and death receptor activated apoptotic pathways in the bursal of Fabricius (BF) cells in the broilers exposed to AFB1 diet. We found that (1) AFB1 diet induced morphological changes in the BF. (2) FCM and TUNEL methods showed that excessive apoptosis could be resulted from AFB1 intake. (3) AFB1-induced apoptosis of bursal cells involved mitochondrial pathway (increase of Bax, Bak, cytC, caspase-9, Apaf-1, caspase-3 and decrease of Bcl-2 and Bcl-xL) and ER pathway (increase of Grp78/Bip, Grp94 and CaM). (4) Oxidative stress was confirmed in the BF of chicken fed on AFB1 diet. Overall, this work is the first to demonstrate that the activation of mitochondria and ER apoptosis pathways can lead to excessive apoptosis in BF cells, and oxidative stress is a crucial driver during AFB1 exposure. PMID:27542244

  9. Granzyme H induces cell death primarily via a Bcl-2-sensitive mitochondrial cell death pathway that does not require direct Bid activation.

    PubMed

    Ewen, Catherine L; Kane, Kevin P; Bleackley, R Chris

    2013-07-01

    Natural killer and T cell-mediated cytotoxicity is important for the elimination of viruses and transformed cells. The granule lytic pathway utilizes perforin and granzymes to induce cell death, while receptor-mediated lytic pathways rely on molecules such as FasL. Pro-apoptotic activities of Granzyme B (GrB) and Fas are well-established, and many of their cellular targets have been identified. However, humans express additional related granzymes - GrA, GrM, GrK, and GrH. Neither the cytotoxic potential of GrH, nor the mechanism by which GrH may induce target cell death is currently understood. We proposed that GrH would have pro-apoptotic activity that would be distinct from that of GrB and FasL, which could be relevant when Fas/FasL or GrB activity or death pathways were impaired. Our results, using a purified recombinant form of GrH, revealed that GrH induced cell death via a Bcl-2-sensitive mitochondrial pathway without direct processing of Bid. Additionally, neither the apoptosome nor caspase-3 was essential to the induction of GrH-mediated cell death. However, GrH did directly process DFF45, potentially leading to DNA damage. Our findings support the idea that multiple, non-redundant death pathways may be initiated by cytotoxic cells to counteract various immune evasion strategies.

  10. Chloroacetic acid induced neuronal cells death through oxidative stress-mediated p38-MAPK activation pathway regulated mitochondria-dependent apoptotic signals.

    PubMed

    Chen, Chun-Hung; Chen, Sz-Jie; Su, Chin-Chuan; Yen, Cheng-Chieh; Tseng, To-Jung; Jinn, Tzyy-Rong; Tang, Feng-Cheng; Chen, Kuo-Liang; Su, Yi-Chang; Lee, kuan-I; Hung, Dong-Zong; Huang, Chun-Fa

    2013-01-07

    Chloroacetic acid (CA), a toxic chlorinated analog of acetic acid, is widely used in chemical industries as an herbicide, detergent, and disinfectant, and chemical intermediates that are formed during the synthesis of various products. In addition, CA has been found as a by-product of chlorination disinfection of drinking water. However, there is little known about neurotoxic injuries of CA on the mammalian, the toxic effects and molecular mechanisms of CA-induced neuronal cell injury are mostly unknown. In this study, we examined the cytotoxicity of CA on cultured Neuro-2a cells and investigated the possible mechanisms of CA-induced neurotoxicity. Treatment of Neuro-2a cells with CA significantly reduced the number of viable cells (in a dose-dependent manner with a range from 0.1 to 3mM), increased the generation of ROS, and reduced the intracellular levels of glutathione depletion. CA also increased the number of sub-G1 hypodiploid cells; increased mitochondrial dysfunction (loss of MMP, cytochrome c release, and accompanied by Bcl-2 and Mcl-1 down-regulation and Bax up-regulation), and activated the caspase cascades activations, which displayed features of mitochondria-dependent apoptosis pathway. These CA-induced apoptosis-related signals were markedly prevented by the antioxidant N-acetylcysteine (NAC). Moreover, CA activated the JNK and p38-MAPK pathways, but did not that ERK1/2 pathway, in treated Neuro-2a cells. Pretreatment with NAC and specific p38-MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125) effectively abrogated the phosphorylation of p38-MAPK and attenuated the apoptotic signals (including: decrease in cytotoxicity, caspase-3/-7 activation, the cytosolic cytochrome c release, and the reversed alteration of Bcl-2 and Bax mRNA) in CA-treated Neuro-2a cells. Taken together, these data suggest that oxidative stress-induced p38-MAPK activated pathway-regulated mitochondria-dependent apoptosis plays an important role in CA-caused neuronal cell

  11. Cardiac arrest triggers hippocampal neuronal death through autophagic and apoptotic pathways

    PubMed Central

    Cui, Derong; Shang, Hanbing; Zhang, Xiaoli; Jiang, Wei; Jia, Xiaofeng

    2016-01-01

    The mechanism of neuronal death induced by ischemic injury remains unknown. We investigated whether autophagy and p53 signaling played a role in the apoptosis of hippocampal neurons following global cerebral ischemia-reperfusion (I/R) injury, in a rat model of 8-min asphyxial cardiac arrest (CA) and resuscitation. Increased autophagosome numbers, expression of lysosomal cathepsin B, cathepsin D, Beclin-1, and microtubule-associated protein light chain 3 (LC3) suggested autophagy in hippocampal cells. The expression of tumor suppressor protein 53 (p53) and its target genes: Bax, p53-upregulated modulator of apoptosis (PUMA), and damage-regulated autophagy modulator (DRAM) were upregulated following CA. The p53-specific inhibitor pifithrin-α (PFT-α) significantly reduced the expression of pro-apoptotic proteins (Bax and PUMA) and autophagic proteins (LC3-II and DRAM) that generally increase following CA. PFT-α also reduced hippocampal neuronal damage following CA. Similarly, 3-methyladenine (3-MA), which inhibits autophagy and bafilomycin A1 (BFA), which inhibits lysosomes, significantly inhibited hippocampal neuronal damage after CA. These results indicate that CA affects both autophagy and apoptosis, partially mediated by p53. Autophagy plays a significant role in hippocampal neuronal death induced by cerebral I/R following asphyxial-CA. PMID:27273382

  12. Decreased Expression of SRSF2 Splicing Factor Inhibits Apoptotic Pathways in Renal Cancer

    PubMed Central

    Kędzierska, Hanna; Popławski, Piotr; Hoser, Grażyna; Rybicka, Beata; Rodzik, Katarzyna; Sokół, Elżbieta; Bogusławska, Joanna; Tański, Zbigniew; Fogtman, Anna; Koblowska, Marta; Piekiełko-Witkowska, Agnieszka

    2016-01-01

    Serine and arginine rich splicing factor 2(SRSF2) belongs to the serine/arginine (SR)-rich family of proteins that regulate alternative splicing. Previous studies suggested that SRSF2 can contribute to carcinogenic processes. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, highly aggressive and difficult to treat, mainly due to resistance to apoptosis. In this study we hypothesized that SRSF2 contributes to the regulation of apoptosis in ccRCC. Using tissue samples obtained from ccRCC patients, as well as independent validation on The Cancer Genome Atlas (TCGA) data, we demonstrate for the first time that expression of SRSF2 is decreased in ccRCC tumours when compared to non-tumorous control tissues. Furthermore, by employing a panel of ccRCC-derived cell lines with silenced SRSF2 expression and qPCR arrays we show that SRSF2 contributes not only to splicing patterns but also to expression of multiple apoptotic genes, including new SRSF2 targets: DIABLO, BIRC5/survivin, TRAIL, BIM, MCL1, TNFRSF9, TNFRSF1B, CRADD, BCL2L2, BCL2A1, and TP53. We also identified a new splice variant of CFLAR, an inhibitor of caspase activity. These changes culminate in diminished caspase-9 activity and inhibition of apoptosis. In summary, we show for the first time that decreased expression of SRSF2 in ccRCC contributes to protection of cancer cells viability. PMID:27690003

  13. β-Amyloid-evoked apoptotic cell death is mediated through MKK6-p66shc pathway.

    PubMed

    Bashir, Muneesa; Parray, Arif A; Baba, Rafia A; Bhat, Hina F; Bhat, Sehar S; Mushtaq, Umar; Andrabi, Khurshid I; Khanday, Firdous A

    2014-03-01

    We have previously shown the involvement of p66shc in mediating apoptosis. Here, we demonstrate the novel mechanism of β-Amyloid-induced toxicity in the mammalian cells. β-Amyloid leads to the phosphorylation of p66shc at the serine 36 residue and activates MKK6, by mediating the phosphorylation at serine 207 residue. Treatment of cells with antioxidants blocks β-Amyloid-induced serine phosphorylation of MKK6, reactive oxygen species (ROS) generation, and hence protected cells against β-Amyloid-induced cell death. Our results indicate that serine phosphorylation of p66shc is carried out by active MKK6. MKK6 knock-down resulted in decreased serine 36 phosphorylation of p66shc. Co-immunoprecipitation results demonstrate a direct physical association between p66shc and WT MKK6, but not with its mutants. Increase in β-Amyloid-induced ROS production was observed in the presence of MKK6 and p66shc, when compared to triple mutant of MKK6 (inactive) and S36 mutant of p66shc. ROS scavengers and knock-down against p66shc, and MKK6 significantly decreased the endogenous level of active p66shc, ROS production, and cell death. Finally, we show that the MKK6-p66shc complex mediates β-Amyloid-evoked apoptotic cell death.

  14. A Biophysical Systems Approach to Identifying the Pathways of Acute and Chronic Doxorubicin Mitochondrial Cardiotoxicity

    PubMed Central

    de Oliveira, Bernardo L.; Niederer, Steven

    2016-01-01

    The clinical use of the anthracycline doxorubicin is limited by its cardiotoxicity which is associated with mitochondrial dysfunction. Redox cycling, mitochondrial DNA damage and electron transport chain inhibition have been identified as potential mechanisms of toxicity. However, the relative roles of each of these proposed mechanisms are still not fully understood. The purpose of this study is to identify which of these pathways independently or in combination are responsible for doxorubicin toxicity. A state of the art mathematical model of the mitochondria including the citric acid cycle, electron transport chain and ROS production and scavenging systems was extended by incorporating a novel representation for mitochondrial DNA damage and repair. In silico experiments were performed to quantify the contributions of each of the toxicity mechanisms to mitochondrial dysfunction during the acute and chronic stages of toxicity. Simulations predict that redox cycling has a minor role in doxorubicin cardiotoxicity. Electron transport chain inhibition is the main pathway for acute toxicity for supratherapeutic doses, being lethal at mitochondrial concentrations higher than 200μM. Direct mitochondrial DNA damage is the principal pathway of chronic cardiotoxicity for therapeutic doses, leading to a progressive and irreversible long term mitochondrial dysfunction. PMID:27870850

  15. Dietary fat modifies mitochondrial and plasma membrane apoptotic signaling in skeletal muscle of calorie-restricted mice.

    PubMed

    López-Domínguez, José Alberto; Khraiwesh, Husam; González-Reyes, José Antonio; López-Lluch, Guillermo; Navas, Plácido; Ramsey, Jon Jay; de Cabo, Rafael; Burón, María Isabel; Villalba, José M

    2013-12-01

    Calorie restriction decreases skeletal muscle apoptosis, and this phenomenon has been mechanistically linked to its protective action against sarcopenia of aging. Alterations in lipid composition of membranes have been related with the beneficial effects of calorie restriction. However, no study has been designed to date to elucidate if different dietary fat sources with calorie restriction modify apoptotic signaling in skeletal muscle. We show that a 6-month calorie restriction decreased the activity of the plasma membrane neutral sphingomyelinase, although caspase-8/10 activity was not altered, in young adult mice. Lipid hydroperoxides, Bax levels, and cytochrome c and AIF release/accumulation into the cytosol were also decreased, although caspase-9 activity was unchanged. No alterations in caspase-3 and apoptotic index (DNA fragmentation) were observed, but calorie restriction improved structural features of gastrocnemius fibers by increasing cross-sectional area and decreasing circularity of fibers in cross sections. Changing dietary fat with calorie restriction produced substantial alterations of apoptotic signaling. Fish oil augmented the protective effect of calorie restriction decreasing plasma membrane neutral sphingomyelinase, Bax levels, caspase-8/10, and -9 activities, while increasing levels of the antioxidant coenzyme Q at the plasma membrane, and potentiating the increase of cross-sectional area and the decrease of fiber circularity in cross sections. Many of these changes were not found when we used lard. Our data support that dietary fish oil with calorie restriction produces a cellular anti-apoptotic environment in skeletal muscle with a downregulation of components involved in the initial stages of apoptosis engagement, both at the plasma membrane and the mitochondria.

  16. Induction of apoptosis by the tropical seaweed Pylaiella littoralis in HT-29 cells via the mitochondrial and MAPK pathways

    NASA Astrophysics Data System (ADS)

    Ye, Bo-Ram; Kim, Junseong; Kim, Min-Sun; Jang, Jiyi; Oh, Chulhong; Kang, Do-Hyung; Qian, Zhong-Ji; Jung, Won-Kyo; Choi, Il-Whan; Heo, Soo-Jin

    2013-12-01

    We demonstrated that an extract from Pylaiella littoralis, collected from the Federate States of Micronesia (FSM), could inhibit the proliferation of tumor cells. P. littoralis extract (PLE) showed anti-proliferative activities in the tumorigenic cells tested, ranging from 20.2% to 67.9%. The highest inhibitory activity, in HT-29 cells, was selected for further experiments. PLE showed no cytotoxic effect in normal cells and inhibited the growth of HT-29 cells depending on concentration and incubation time. PLE-treated HT-29 cells showed the typical morphological characteristics of apoptosis, such as apoptotic body formation and DNA fragmentation. PLE also induced mitochondrial membrane potential depolarization and resulted in increased mitochondrial membrane permeability, compared with untreated cells. PLE decreased Bcl-2 protein and increased Bax protein expression, activating caspase-3 and poly (ADP-ribose) polymerase (PARP) expression via the caspase pathway. PLE also increased the phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), and it reduced cell viability in treatment cells with specific inhibitors such as PD98059 (a specific inhibitor of ERK), SP600125 (a specific inbibitor of JNK), and SB 203580 (a specific inbibitor of p38 MAPK). via the the mitogen-activated protein kinases (MAPKs) pathway. These results suggest that PLE inhibits the proliferation of HT-29 cells by affecting the caspase and MAPK pathways involved in the induction of apoptosis. Thus, we suggest that P. littoralis extract might be potential candidate agents for the treatment of human colorectal cancer.

  17. Mitochondrial targeting of α-tocopheryl succinate enhances its pro-apoptotic efficacy: a new paradigm for effective cancer therapy.

    PubMed

    Dong, Lan-Feng; Jameson, Victoria J A; Tilly, David; Prochazka, Lubomir; Rohlena, Jakub; Valis, Karel; Truksa, Jaroslav; Zobalova, Renata; Mahdavian, Elahe; Kluckova, Katarina; Stantic, Marina; Stursa, Jan; Freeman, Ruth; Witting, Paul K; Norberg, Erik; Goodwin, Jacob; Salvatore, Brian A; Novotna, Jana; Turanek, Jaroslav; Ledvina, Miroslav; Hozak, Pavel; Zhivotovsky, Boris; Coster, Mark J; Ralph, Stephen J; Smith, Robin A J; Neuzil, Jiri

    2011-06-01

    Mitochondria are emerging as intriguing targets for anti-cancer agents. We tested here a novel approach, whereby the mitochondrially targeted delivery of anti-cancer drugs is enhanced by the addition of a triphenylphosphonium group (TPP(+)). A mitochondrially targeted analog of vitamin E succinate (MitoVES), modified by tagging the parental compound with TPP(+), induced considerably more robust apoptosis in cancer cells with a 1-2 log gain in anti-cancer activity compared to the unmodified counterpart, while maintaining selectivity for malignant cells. This is because MitoVES associates with mitochondria and causes fast generation of reactive oxygen species that then trigger mitochondria-dependent apoptosis, involving transcriptional modulation of the Bcl-2 family proteins. MitoVES proved superior in suppression of experimental tumors compared to the untargeted analog. We propose that mitochondrially targeted delivery of anti-cancer agents offers a new paradigm for increasing the efficacy of compounds with anti-cancer activity.

  18. Droxinostat, a Histone Deacetylase Inhibitor, Induces Apoptosis in Hepatocellular Carcinoma Cell Lines via Activation of the Mitochondrial Pathway and Downregulation of FLIP1

    PubMed Central

    Liu, Jing; Li, Guangming; Wang, Xiang; Wang, Liang; Zhao, Rui; Wang, Juanxia; Kong, Yin; Ding, Jie; Li, Juan; Zhang, Lingyi

    2016-01-01

    Background: The current chemotherapeutic outcomes for hepatocellular carcinoma (HCC) are not encouraging, and long-term survival of this patient group remains poor. Recent studies have demonstrated the utility of histone deacetylase inhibitors that can disrupt cell proliferation and survival in HCC management. However, the effects of droxinostat, a type of histone deacetylase inhibitor, on HCC remain to be established. Methods: The effects of droxinostat on HCC cell lines SMMC-7721 and HepG2 were investigated. Histone acetylation and apoptosis-modulating proteins were assessed via Western blot. Proliferation was examined with 3-(4, 5 dimetyl-2-thiazolyl)-2, 5-diphenyl 2H-tetrazolium bromide, cell proliferation, and real-time cell viability assays, and apoptosis with flow cytometry. Results: Droxinostat inhibited proliferation and colony formation of the HCC cell lines examined. Hepatoma cell death was induced through activation of the mitochondrial apoptotic pathway and downregulation of FLIP expression. Droxinostat suppressed histone deacetylase (HDAC) 3 expression and promoted acetylation of histones H3 and H4. Knockdown of HDAC3 induced hepatoma cell apoptosis and histone H3 and H4 acetylation. Conclusions: Droxinostat suppresses HDAC3 expression and induces histone acetylation and HCC cell death through activation of the mitochondrial apoptotic pathway and downregulation of FLIP, supporting its potential application in the treatment of HCC. PMID:26947884

  19. The anesthetic agent sevoflurane attenuates pulmonary acute lung injury by modulating apoptotic pathways

    PubMed Central

    Wang, L.; Ye, Y.; Su, H.B.; Yang, J.P.

    2017-01-01

    The objective of this study was to evaluate lung protection by the volatile anesthetic sevoflurane (SEVO), which inhibits apoptosis. Male Sprague-Dawley rats (250–280 g; n=18) were randomly divided into three groups. The LPS group received 5 mg/kg endotoxin (lipopolysaccharide), which induced acute lung injury (ALI). The control (CTRL) group received normal saline and the SEVO group received sevoflurane (2.5%) for 30 min after ALI was induced by 5 mg/kg LPS. Samples were collected for analysis 12 h after LPS. Lung injury was assessed by pathological observations and tissue wet to dry weight (W/D) ratios. Apoptotic index (AI) was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and electron microscopy. Caspase-3 and cleaved-caspase-3 protein levels were determined by immunocytochemistry and western blotting, respectively. Bcl-xl levels were measured by western blotting and Bcl-2 levels by quantitative real-time polymerase chain reaction and western blotting. In the LPS group, W/D ratios, AI values, caspase-3 and cleaved-caspase-3 levels were significantly higher than in the CTRL group and lung injury was more severe. In the SEVO group, W/D ratios, AI, caspase-3 and cleaved-caspase-3 were lower than in the LPS group. Bcl-2 and Bcl-xl expression were higher than in the LPS group and lung injury was attenuated. Sevoflurane inhalation protected the lungs from injury by regulating caspase-3 activation and Bcl-xl and Bcl-2 expression to inhibit excessive cell apoptosis, and such apoptosis might be important in the pathogenesis of LPS-induced ALI. PMID:28225890

  20. Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells

    PubMed Central

    ZHU, YUE-YONG; HUANG, HONG-YAN; WU, YIN-LIAN

    2015-01-01

    Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine-123 DNA-binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose-dependent, as well as time-dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub-G1 (apoptotic) phase of the cell cycle, in a dose-dependent manner. Staining with Annexin V-fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose-dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose-dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC. PMID:26151733

  1. PTEN regulates apoptotic cell death through PI3-K/Akt/GSK3β signaling pathway in DMH induced early colon carcinogenesis in rat.

    PubMed

    Saini, Manpreet Kaur; Sanyal, Sankar Nath

    2012-08-01

    Phosphatidylinositol 3-kinase (PI3-K) and Akt (protein kinase B), are both essential signaling molecules that are up-regulated in various cancers. Here, we examined the molecular mechanisms by which PI3-K and Akt expression are regulated by glycogen synthase kinase-3β (GSK-3β) and the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the early stages of experimental colon carcinogenesis. 1,2-dimethylhydrazine (DMH) was utilized for the induction of colon cancer while piroxicam, a traditional non-steroidal anti-inflammatory drug and c-phycocyanin, a biliprotein from Spirulina platensis (cyanobacterium) as the chemopreventive agents. Western blotting and immunofluorescence results indicated that the expression of PI3-K and Akt was promoted in the DMH group while least apoptosis was detected in this group as analyzed by Hoechst 33342-propidium iodide co-staining. DMH group further detected lower GSK-3β and PTEN expression as compared to other groups. Piroxicam and c-phycocyanin treatment resulted significant apoptotic cell death while showing low PI3-K and Akt expressions. Mitochondrial membrane potential (ΔΨ(M)) alterations (examined by JC-1 and rhodamine 123 labeling of colonocytes) and fluorescence intensity measurement of ROS level, were also analyzed showing the raised ΔΨ(M) while reduced ROS levels in DMH group, however piroxicam and c-phycocyanin treatment resulted in falling of ΔΨ(M) although both stimulated the ROS production as analyzed by flow cytometry. The present study thus identified that piroxicam, a traditional NSAID and c-phycocyanin, a newly discovered COX-2 selective inhibitor, constitute remarkable chemopreventive targets in mediating apoptosis in the DMH induced early rat colon carcinogenesis via regulating PI3-K/Akt/GSK-3β/PTEN signaling pathways. Further, a combination of the two drugs provides a better therapeutic option, than the monotherapy regimen.

  2. Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling

    PubMed Central

    Otera, Hidenori; Miyata, Non; Kuge, Osamu

    2016-01-01

    Mitochondrial fission facilitates cytochrome c release from the intracristae space into the cytoplasm during intrinsic apoptosis, although how the mitochondrial fission factor Drp1 and its mitochondrial receptors Mff, MiD49, and MiD51 are involved in this reaction remains elusive. Here, we analyzed the functional division of these receptors with their knockout (KO) cell lines. In marked contrast to Mff-KO cells, MiD49/MiD51-KO and Drp1-KO cells completely resisted cristae remodeling and cytochrome c release during apoptosis. This phenotype in MiD49/51-KO cells, but not Drp1-KO cells, was completely abolished by treatments disrupting cristae structure such as OPA1 depletion. Unexpectedly, OPA1 oligomers generally thought to resist cytochrome c release by stabilizing the cristae structure were similarly disassembled in Drp1-KO and MiD49/51-KO cells, indicating that disassembly of OPA1 oligomers is not directly linked to cristae remodeling for cytochrome c release. Together, these results indicate that Drp1-dependent mitochondrial fission through MiD49/MiD51 regulates cristae remodeling during intrinsic apoptosis. PMID:26903540

  3. Apoptotic effects of Physalis minima L. chloroform extract in human breast carcinoma T-47D cells mediated by c-myc-, p53-, and caspase-3-dependent pathways.

    PubMed

    Ooi, Kheng Leong; Tengku Muhammad, Tengku Sifzizul; Lim, Chui Hun; Sulaiman, Shaida Fariza

    2010-03-01

    The chloroform extract of Physalis minima produced a significant growth inhibition against human T-47D breast carcinoma cells as compared with other extracts with an EC(50) value of 3.8 microg/mL. An analysis of cell death mechanisms indicated that the extract elicited an apoptotic cell death. mRNA expression analysis revealed the coregulation of apoptotic genes, that is, c-myc , p53, and caspase-3. The c-myc was significantly induced by the chloroform extract at the earlier phase of treatment, followed by p53 and caspase-3. Biochemical assay and ultrastructural observation displayed typical apoptotic features in the treated cells, including DNA fragmentation, blebbing and convolution of cell membrane, clumping and margination of chromatin, and production of membrane-bound apoptotic bodies. The presence of different stages of apoptotic cell death and phosphatidylserine externalization were further reconfirmed by annexin V and propidium iodide staining. Thus, the results from this study strongly suggest that the chloroform extract of P. minima induced apoptotic cell death via p53-, caspase-3-, and c-myc-dependent pathways.

  4. HnRNP-L mediates bladder cancer progression by inhibiting apoptotic signaling and enhancing MAPK signaling pathways.

    PubMed

    Lv, Daojun; Wu, Huayan; Xing, Rongwei; Shu, Fangpeng; Lei, Bin; Lei, Chengyong; Zhou, Xumin; Wan, Bo; Yang, Yu; Zhong, Liren; Mao, Xiangming; Zou, Yaguang

    2017-01-11

    Heterogeneous nuclear ribonucleoprotein L (hnRNP-L) is a promoter of various kinds of cancers, but its actions in bladder cancer (BC) are unclear. In this study, we investigated the function and the underlying mechanism of hnRNP-L in bladder carcinogenesis. Our results demonstrated that enhanced hnRNP-L expression in BC tissues was associated with poor overall survival of BC patients. Depletion of hnRNP-L significantly suppressed cell proliferation in vitro and inhibited xenograft tumor growth in vivo. Furthermore, downregulation of hnRNP-L resulted in G1-phase cell cycle arrest and enhanced apoptosis accompanied by inhibition of EMT and cell migration. All these cellular changes were reversed by ectopic expression of hnRNP-L. Deletion of hnRNP-L resulted in decreased expression of Bcl-2, enhanced expression of caspases-3, -6 and -9 and inhibition of the MAPK signaling pathway. These findings demonstrate that hnRNP-L contributes to poor prognosis and tumor progression of BC by inhibiting the intrinsic apoptotic signaling and enhancing MAPK signaling pathways.

  5. The miR-204-3p-targeted IGFBP2 pathway is involved in xanthohumol-induced glioma cell apoptotic death.

    PubMed

    Chen, Peng-Hsu; Chang, Cheng-Kuei; Shih, Chwen-Ming; Cheng, Chia-Hsiung; Lin, Cheng-Wei; Lee, Chin-Cheng; Liu, Ann-Jeng; Ho, Kuo-Hao; Chen, Ku-Chung

    2016-11-01

    Xanthohumol (XN), a prenylated chalcone extracted from hop plant Humulus lupulus L. (Cannabaceae), has potential for cancer therapy, including gliomas. Micro (mi)RNAs are small noncoding RNAs that control gene expression. Several miRNAs have been identified to participate in regulating glioma development. However, no studies have demonstrated whether miRNA is involved in XN cytotoxicity resulting in glioma cell death. This study investigated the effects of XN-mediated miRNA expression in activating apoptotic pathways in glioblastoma U87 MG cells. First, we found that XN significantly reduced cell viability and induced apoptosis via pro-caspase-3/8 cleavage and poly(ADP ribose) polymerase (PARP) degradation. We also identified that pro-caspase-9 cleavage, Bcl2 family expression changes, mitochondrial dysfunction, and intracellular ROS generation also participated in XN-induced glioma cell death. With a microarray analysis, miR-204-3p was identified as the most upregulated miRNA induced by XN cytotoxicity. The extracellular signal-regulated kinase (ERK)/c-Fos pathway was validated to participate in XN-upregulated miR-204-3p expression. With a promoter assay and ChIP analysis, we found that c-Fos dose-dependently bound to the miR-204-3p gene promoter region. Furthermore, miR-204-3p levels decreased in several glioma cell lines compared to astrocytes. Overexpression of miR-204-3p enhanced glioma cell apoptosis. IGFBP2, an upregulated regulator of glioma proliferation, was validated by a TCGA analysis as a direct target gene of miR-204-3p. XN's inhibition of the IGFBP2/AKT/Bcl2 pathway via miR-204-3p targeting played a critical role in mediating glioma cell death. These results emphasized that the XN-mediated miR-204-3p network may provide novel therapeutic strategies for future glioblastoma therapy and drug development.

  6. Glaucarubinone sensitizes KB cells to paclitaxel by inhibiting ABC transporters via ROS-dependent and p53-mediated activation of apoptotic signaling pathways

    PubMed Central

    Karthikeyan, Subburayan; Hoti, Sugeerappa Laxmanappa; Nazeer, Yasin; Hegde, Harsha Vasudev

    2016-01-01

    Multidrug resistance (MDR) is considered to be the major contributor to failure of chemotherapy in oral squamous cell carcinoma (SCC). This study was aimed to explore the effects and mechanisms of glaucarubinone (GLU), one of the major quassinoids from Simarouba glauca DC, in potentiating cytotoxicity of paclitaxel (PTX), an anticancer drug in KB cells. Our data showed that the administration of GLU pre-treatment significantly enhanced PTX anti-proliferative effect in ABCB1 over-expressing KB cells. The Rh 123 drug efflux studies revealed that there was a significant transport function inhibition by GLU-PTX treatment. Interestingly, it was also found that this enhanced anticancer efficacy of GLU was associated with PTX-induced cell arrest in the G2/M phase of cell cycle. Further, the combined treatment of GLU-PTX had significant decrease in the expression levels of P-gp, MRPs, and BCRP in resistant KB cells at both mRNA and protein levels. Furthermore, the combination treatments showed significant reactive oxygen species (ROS) production, chromatin condensation and reduced mitochondrial membrane potential in resistant KB cells. The results from DNA fragmentation analysis also demonstrated the GLU induced apoptosis in KB cells and its synergy with PTX. Importantly, GLU and/or PTX triggered apoptosis through the activation of pro-apoptotic proteins such as p53, Bax, and caspase-9. Our findings demonstrated for the first time that GLU causes cell death in human oral cancer cells via the ROS-dependent suppression of MDR transporters and p53-mediated activation of the intrinsic mitochondrial pathway of apoptosis. Additionally, the present study also focussed on investigation of the protective effect of GLU and combination drugs in human normal blood lymphocytes. Normal blood lymphocytes assay indicated that GLU is able to induce selective toxicity in cancer cells and in silico molecular docking studies support the choice of GLU as ABC inhibitor to enhance PTX efficacy

  7. Distinct muscle apoptotic pathways are activated in muscles with different fiber types a rat model of critical illness myopathy

    PubMed Central

    Barnes, Benjamin T.; Confides, Amy L.; Rich, Mark M.; Dupont-Versteegden, Esther E.

    2015-01-01

    Critical illness myopathy (CIM) is associated with severe muscle atrophy and fatigue in affected patients. Apoptotic signaling is involved in atrophy and is elevated in muscles from patients with CIM. In this study we investigated underlying mechanisms of apoptosis-related pathways in muscles with different fiber type composition in a rat model of CIM using denervation and glucocorticoid administration (denervation and steroid-induced myopathy, DSIM). Soleus and tibialis anterior (TA) muscles showed severe muscle atrophy (40–60% of control muscle weight) and significant apoptosis in interstitial as well as myofiber nuclei that was similar between the two muscles with DSIM. Caspase-3 and −8 activities, but not caspase-9 and −12, were elevated in TA and not in soleus muscle, while the caspase-independent proteins endonuclease G (EndoG) and apoptosis inducing factor (AIF) were not changed in abundance nor differentially localized in either muscle. Anti-apoptotic proteins HSP70, −27, and apoptosis repressor with a caspase recruitment domain (ARC) were elevated in soleus compared to TA muscle and ARC was significantly decreased with induction of DSIM in soleus. Results indicate that apoptosis is a significant process associated with DSIM in both soleus and TA muscles, and that apoptosis-associated processes are differentially regulated in muscles of different function and fiber type undergoing atrophy due to DSIM. We conclude that interventions combating apoptosis with CIM may need to be directed towards inhibiting caspase-dependent as well as -independent mechanisms to be able to affect muscles of all fiber types. PMID:25740800

  8. Targeting of apoptotic pathways by SMAC or BH3 mimetics distinctly sensitizes paclitaxel-resistant triple negative breast cancer cells.

    PubMed

    Panayotopoulou, Effrosini G; Müller, Anna-Katharina; Börries, Melanie; Busch, Hauke; Hu, Guohong; Lev, Sima

    2017-02-06

    Standard chemotherapy is the only systemic treatment for triple-negative breast cancer (TNBC), and despite the good initial response, resistance remains a major therapeutic obstacle. Here, we employed a High-Throughput Screen to identify targeted therapies that overcome chemoresistance in TNBC. We applied short-term paclitaxel treatment and screened 320 small-molecule inhibitors of known targets to identify drugs that preferentially and efficiently target paclitaxel-treated TNBC cells. Among these compounds the SMAC mimetics (BV6, Birinapant) and BH3-mimetics (ABT-737/263) were recognized as potent targeted therapy for multiple paclitaxel-residual TNBC cell lines. However, acquired paclitaxel resistance through repeated paclitaxel pulses result in desensitization to BV6, but not to ABT-263, suggesting that short- and long-term paclitaxel resistance are mediated by distinct mechanisms. Gene expression profiling of paclitaxel-residual, -resistant and naïve MDA-MB-231 cells demonstrated that paclitaxel-residual, as opposed to -resistant cells, were characterized by an apoptotic signature, with downregulation of anti-apoptotic genes (BCL2, BIRC5), induction of apoptosis inducers (IL24, PDCD4), and enrichment of TNFα/NF-κB pathway, including upregulation of TNFSF15, coupled with cell-cycle arrest. BIRC5 and FOXM1 downregulation and IL24 induction was also evident in breast cancer patient datasets following taxane treatment. Exposure of naïve or paclitaxel-resistant cells to supernatants of paclitaxel-residual cells sensitized them to BV6, and treatment with TNFα enhanced BV6 potency, suggesting that sensitization to BV6 is mediated, at least partially, by secreted factor(s). Our results suggest that administration of SMAC or BH3 mimetics following short-term paclitaxel treatment could be an effective therapeutic strategy for TNBC, while only BH3-mimetics could effectively overcome long-term paclitaxel resistance.

  9. Distinct muscle apoptotic pathways are activated in muscles with different fiber types in a rat model of critical illness myopathy.

    PubMed

    Barnes, Benjamin T; Confides, Amy L; Rich, Mark M; Dupont-Versteegden, Esther E

    2015-06-01

    Critical illness myopathy (CIM) is associated with severe muscle atrophy and fatigue in affected patients. Apoptotic signaling is involved in atrophy and is elevated in muscles from patients with CIM. In this study we investigated underlying mechanisms of apoptosis-related pathways in muscles with different fiber type composition in a rat model of CIM using denervation and glucocorticoid administration (denervation and steroid-induced myopathy, DSIM). Soleus and tibialis anterior (TA) muscles showed severe muscle atrophy (40-60% of control muscle weight) and significant apoptosis in interstitial as well as myofiber nuclei that was similar between the two muscles with DSIM. Caspase-3 and -8 activities, but not caspase-9 and -12, were elevated in TA and not in soleus muscle, while the caspase-independent proteins endonuclease G (EndoG) and apoptosis inducing factor (AIF) were not changed in abundance nor differentially localized in either muscle. Anti-apoptotic proteins HSP70, -27, and apoptosis repressor with a caspase recruitment domain (ARC) were elevated in soleus compared to TA muscle and ARC was significantly decreased with induction of DSIM in soleus. Results indicate that apoptosis is a significant process associated with DSIM in both soleus and TA muscles, and that apoptosis-associated processes are differentially regulated in muscles of different function and fiber type undergoing atrophy due to DSIM. We conclude that interventions combating apoptosis with CIM may need to be directed towards inhibiting caspase-dependent as well as -independent mechanisms to be able to affect muscles of all fiber types.

  10. Respiratory Substrates Regulate S-Nitrosylation of Mitochondrial Proteins through a Thiol-Dependent Pathway

    PubMed Central

    2015-01-01

    S-Nitrosylation is a reversible post-translational modification on cysteinyl thiols that can modulate the function of redox-sensitive proteins. The S-nitrosylation of mitochondrial proteins has been shown to regulate various mitochondrial activities involved in energy-transducing systems and mitochondrion-driven apoptosis. In isolated rat brain mitochondria, we demonstrate that mitochondrial protein S-nitrosylation is regulated by respiratory substrates (glutamate/malate) through a thiol-dependent pathway. Mitochondrial proteins become susceptible to S-nitrosoglutathione (GSNO)-induced S-nitrosylation in mitochondria with an oxidized environment (low glutathione (GSH), NADH, and NADPH, and high GSSG, NAD+, and NADP+) caused by isolation of mitochondria using a discontinuous Percoll gradient. Activation of mitochondrial respiration by respiratory substrates leads to increased NAD(P)H and GSH levels, which in turn reduces mitochondrial S-nitrosylated proteins. 1-Chloro-2,4-dinitrobenzene (CDNB), which depletes mitochondrial GSH and inhibits the thioredoxin–thioredoxin reductase system, prevented the denitrosylation of mitochondrial proteins caused by respiratory substrate treatment. Using biotin-switch coupled with LC-MS/MS, several mitochondrial proteins were identified as targets of S-nitrosylation including adenine nucleotide translocase (ANT) and voltage-dependent anion channel (VDAC), important components of the mitochondria permeability transition pore (MPTP), as well as ATP synthase. The S-nitrosylation of ATP synthase by GSNO was found to inhibit its activity. These findings emphasize the importance of respiratory substrates in regulating S-nitrosylation through a thiol-dependent (GSH and/or thioredoxin) pathway, with implications for mitochondrial bioenergetics and mitochondrion-driven apoptosis. PMID:24716714

  11. Mitochondrial-mediated apoptosis pathway in alveolar epithelial cells exposed to the metals in combustion-generated particulate matter.

    PubMed

    Visalli, Giuseppa; Baluce, Barbara; Bertuccio, Maria; Picerno, Isa; Di Pietro, Angela

    2015-01-01

    Previously a significant mitochondrial impairment was identified in alveolar epithelial cells exposed to metals adsorbed to combustion-generated particulate matter (PM). Due to the critical role of mitochondria in apoptosis, the aim of this study was to investigate the pro-apoptotic potential of metals present in oil fly ash (OFA). A549 cells were exposed to water-soluble components of an OFA sample, containing vanadium [V(IV)], iron [Fe(III)], and nickel [Ni(II)] (68.8, 110.4, and 18 μM, respectively). Experiments were also performed using individual metal solutions. Apoptosis was detected and the mitochondrial role was assessed by a caspase-9 inhibitor (Z-LEHD-FMK). To determine whether the presence of impaired mitochondria in unexposed daughter cells increased apoptosis, an in vitro model was developed that allowed determination of effects until the third cell generation. To specifically examine the toxicity of vanadium (V), that characterize the airborne pollutant examined in this study, p53involvement and metabolic impairment through changes in HIF-1α and Glut-1 expression were determined. OFA and individual metal solutions produced significant apoptosis in the progeny of exposed cells, triggering the intrinsic apoptosis pathway. In apoptosis induced by poorly genotoxic metal V, p53 did not play a significant role. However, V exposure increased nuclear translocation of HIF-1α and expression of the Glut-1 receptor, indicating metabolic impairment due to metal-induced mitochondrial dysfunction. Overall, these results improve our knowledge of the pathogenic role that airborne metals and in particular V exerted in respiratory epithelium.

  12. Hexane extract of Raphanus sativus L. roots inhibits cell proliferation and induces apoptosis in human cancer cells by modulating genes related to apoptotic pathway.

    PubMed

    Beevi, Syed Sultan; Mangamoori, Lakshmi Narasu; Subathra, Murugan; Edula, Jyotheeswara Reddy

    2010-09-01

    Raphanus sativus, a common cruciferous vegetable has been attributed to possess a number of pharmacological and therapeutic properties. It has been used in indigenous system of medicine for the treatment of various human ailments in India. This present study evaluated the chemopreventive efficacy of different parts of R. sativus such as root, stem and leaves, extracted with solvents of varying polarity and investigated the molecular mechanism leading to growth arrest and apoptotic cell death in human cancer cell lines. Of the different parts, significant growth inhibitory effect was observed with hexane extract of R. sativus root. Analysis of hexane extract by GC-MS revealed the presence of several isothiocyanates (ITCs) such as 4-(methylthio)-3-butenyl isothiocyanate (MTBITC), 4-(methylthio)-3-butyl isothiocyanate (erucin), 4-methylpentyl isothiocyanate, 4-pentenyl isothiocyanate and sulforaphene. R. sativus root extract induced cell death both in p53 proficient and p53 deficient cell lines through induction of apoptotic signaling pathway regardless of the p53 status of cells. The molecular mechanisms underlying R. sativus-induced apoptosis may involve interactions among Bcl(2) family genes, as evidenced by up-regulation of pro-apoptotic genes and down-regulation of anti-apoptotic genes along with activation of Caspase-3. Our findings present the first evidence that hexane extract of R. sativus root exerts potential chemopreventive efficacy and induces apoptosis in cancer cell lines through modulation of genes involved in apoptotic signaling pathway.

  13. The Amaryllidaceae isocarbostyril narciclasine induces apoptosis by activation of the death receptor and/or mitochondrial pathways in cancer cells but not in normal fibroblasts.

    PubMed

    Dumont, Patrick; Ingrassia, Laurent; Rouzeau, Sébastien; Ribaucour, Fabrice; Thomas, Stéphanie; Roland, Isabelle; Darro, Francis; Lefranc, Florence; Kiss, Robert

    2007-09-01

    Our study has shown that the Amaryllidaceae isocarbostyril narciclasine induces marked apoptosis-mediated cytotoxic effects in human cancer cells but not in normal fibroblasts by triggering the activation of the initiator caspases of the death receptor pathway (caspase-8 and caspase-10) at least in human MCF-7 breast and PC-3 prostate carcinoma cells. The formation of the Fas and death receptor 4 (DR4) death-inducing signaling complex was clearly evidenced in MCF-7 and PC-3 cancer cells. Caspase-8 was found to interact with Fas and DR4 receptors on narciclasine treatment. However, narciclasine-induced downstream apoptotic pathways in MCF-7 cells diverged from those in PC-3 cells, where caspase-8 directly activated effector caspases such as caspase-3 in the absence of any further release of mitochondrial proapoptotic effectors. In contrast, in MCF-7 cells, the apoptotic process was found to require an amplification step that is mitochondria-dependent, with Bid processing, release of cytochrome c, and caspase-9 activation. It is postulated that the high selectivity of narciclasine to cancer cells might be linked, at least in part, to this activation of the death receptor pathway. Normal human fibroblasts appear approximately 250-fold less sensitive to narciclasine, which does not induce apoptosis in these cells probably due to the absence of death receptor pathway activation.

  14. Hypothyroidism reduces mammary tumor progression via Β-catenin-activated intrinsic apoptotic pathway in rats.

    PubMed

    López Fontana, C M; Zyla, L E; Santiano, F E; Sasso, C V; Cuello-Carrión, F D; Pistone Creydt, V; Fanelli, M A; Carón, R W

    2017-02-13

    Experimental hypothyroidism retards mammary carcinogenesis promoting apoptosis of tumor cells. β-catenin plays a critical role in cell adhesion and intracellular signaling pathways conditioning the prognosis of breast cancer. However, the mechanistic connections associated with the expression of β-catenin in thyroid status and breast cancer are not known. Therefore, we studied the relationship between the expression and localization of β-catenin and apoptosis in mammary tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) in hypothyroid (Hypot) and euthyroid (EUT) rats. Female Sprague Dawley rats were treated with a dose of DMBA (15 mg/rat) at 55 days of age and were then divided into two groups: HypoT (0.01% 6-N-propyl-2-thiouracil in drinking water, n = 54) and EUT (untreated control, n = 43). Latency, incidence and progression of tumors were determined. At sacrifice, tumors were obtained for immunohistological studies and Western Blot. The latency was longer (p < 0.05), the incidence was lower (p < 0.0001) and tumor growth was slower (p < 0.01) in HypoT rats compared to EUT. The expression of Bax, cleaved caspase-9 and caspase-3 was significantly higher in tumors of HypoT than in EUT (p < 0.05) indicating the activation of the intrinsic pathway. In this group, β-catenin was expressed in the plasma membrane and with less intensity, while its expression was nuclear and with greater intensity in the EUT (p < 0.05). Moreover, the expression of survivin was reduced in tumors of HypoT rats (p < 0.05). In conclusion, decreased expression of β-catenin and its normal location in membrane of mammary tumors are associated with augmented apoptosis via activation of the intrinsic pathway in HypoT rats.

  15. Defending the mitochondria: The pathways of mitophagy and mitochondrial-derived vesicles.

    PubMed

    Roberts, Rosalind F; Tang, Matthew Y; Fon, Edward A; Durcan, Thomas M

    2016-10-01

    Mitochondria are the powerhouses for the cell, consuming oxygen to generate sufficient energy for the maintenance of normal cellular processes. However, a deleterious consequence of this process are reactive oxygen species generated as side-products of these reactions. As a means to protect mitochondria from damage, cells and mitochondria have developed a wide-range of mitochondrial quality control mechanisms that remove damaged mitochondrial cargo, enabling the mitochondria to repair the damage and ultimately restore their normal function. If the damage is extensive and mitochondria can no longer be repaired, a process termed mitophagy is initiated in which the mitochondria are directed for autophagic clearance. Canonical mitophagy is regulated by two proteins, PINK1 and Parkin, which are mutated in familial forms of Parkinson's disease. In this review, we discuss recent work elucidating the mechanism of PINK1/Parkin-mediated mitophagy, along with recently uncovered PINK1/Parkin-independent mitophagy pathways. Moreover, we describe a novel mitochondrial quality control pathway, involving mitochondrial-derived vesicles that direct distinct and damaged mitochondrial cargo for degradation in the lysosome. Finally, we discuss the association between mitochondrial quality control, cardiac, hepatic and neurodegenerative disease and discuss the possibility of targeting these pathways for therapeutic purposes.

  16. Partial construction of apoptotic pathway in PBMC obtained from active SLE patients and the significance of plasma TNF-alpha on this pathway.

    PubMed

    Pitidhammabhorn, Dhanesh; Kantachuvesiri, Surasak; Totemchokchyakarn, Kitti; Kitiyanant, Yindee; Ubol, Sukathida

    2006-09-01

    Systemic lupus erythematosus (SLE) is a complex autoimmune disorder that affects various organs and systems. Increased apoptosis, together with defects in the uptake of apoptotic bodies, are thought to have a pathogenic role in SLE. By detection of chromatin condensation, 30% of apoptosis was detected in peripheral blood mononuclear cells (PBMC) from Thai patients with active SLE. Therefore, understanding of the molecular processes in PBMC apoptosis may allow us to gain insight into pathophysiology of SLE. Thus, genes involved in the apoptosis of PBMC from these patients were investigated ex vivo by cDNA array analysis. Seventeen apoptosis-related genes were stimulated in active SLE, more than twofold higher than in inactive SLE. These genes are classified into six groups, namely death receptors, death ligands, caspases, bcl-family, and neutral proteases and genes involved in endoplasmic reticulum stress-mediated apoptosis, such as caspase-4 and GADD153. Among those stimulated genes, tumor necrosis factor (TNF) and the TNF-receptor family were drastically up-regulated 60- and 19-fold higher than in healthy controls, respectively. Moreover, the degree of apoptosis correlated with the level of TNF-alpha in plasma, suggesting that the TNF family plays a role in the induction of apoptosis in SLE. To verify this hypothesis, PBMC from healthy individuals were treated with plasma from active SLE patients in the presence or absence of etanercept, a TNF inhibitor. In the presence of etanercept, active SLE plasma reduced the level of apoptosis to 26.43%. In conclusion, massive apoptotic death of PBMC occurred during the active stage of SLE. The molecular pathway of SLE-PBMC apoptosis was mediated at least via TNF/TNFR signaling pathway, which was confirmed by functional test of TNF-alpha in SLE patients' plasma.

  17. Melatonin mitigates mitochondrial malfunction.

    PubMed

    León, Josefa; Acuña-Castroviejo, Darío; Escames, Germane; Tan, Dun-Xian; Reiter, Russel J

    2005-01-01

    Melatonin, or N-acetyl-5-methoxytryptamine, is a compound derived from tryptophan that is found in all organisms from unicells to vertebrates. This indoleamine may act as a protective agent in disease conditions such as Parkinson's, Alzheimer's, aging, sepsis and other disorders including ischemia/reperfusion. In addition, melatonin has been proposed as a drug for the treatment of cancer. These disorders have in common a dysfunction of the apoptotic program. Thus, while defects which reduce apoptotic processes can exaggerate cancer, neurodegenerative disorders and ischemic conditions are made worse by enhanced apoptosis. The mechanism by which melatonin controls cell death is not entirely known. Recently, mitochondria, which are implicated in the intrinsic pathway of apoptosis, have been identified as a target for melatonin actions. It is known that melatonin scavenges oxygen and nitrogen-based reactants generated in mitochondria. This limits the loss of the intramitochondrial glutathione and lowers mitochondrial protein damage, improving electron transport chain (ETC) activity and reducing mtDNA damage. Melatonin also increases the activity of the complex I and complex IV of the ETC, thereby improving mitochondrial respiration and increasing ATP synthesis under normal and stressful conditions. These effects reflect the ability of melatonin to reduce the harmful reduction in the mitochondrial membrane potential that may trigger mitochondrial transition pore (MTP) opening and the apoptotic cascade. In addition, a reported direct action of melatonin in the control of currents through the MTP opens a new perspective in the understanding of the regulation of apoptotic cell death by the indoleamine.

  18. Daunorubicin induces cell death via activation of apoptotic signalling pathway and inactivation of survival pathway in muscle-derived stem cells.

    PubMed

    Stulpinas, Aurimas; Imbrasaitė, Aušra; Kalvelytė, Audronė Valerija

    2012-04-01

    Daunorubicin (as well as other anthracyclines) is known to be toxic to heart cells and other cells in organism thus limiting its applicability in human cancer therapy. To investigate possible mechanisms of daunorubicin cytotoxicity, we used stem cell lines derived from adult rabbit skeletal muscle. Recently, we have shown that daunorubicin induces apoptotic cell death in our cell model system and distinctly influences the activity of MAP kinases. Here, we demonstrate that two widely accepted antagonistic signalling pathways namely proapoptotic JNK and prosurvival PI3K/AKT participate in apoptosis. Using the Western blot method, we observed the activation of JNK and phosphorylation of its direct target c-Jun along with inactivation of AKT and its direct target GSK in the course of programmed cell death. By means of small-molecule kinase inhibitors and transfection of cells with the genes of the components of these pathways, c-Jun and AKT, we confirm that JNK signalling pathway is proapoptotic, whereas AKT is antiapoptotic in daunorubicin-induced muscle cells. These findings could contribute to new approaches which will result in less toxicity and fewer side effects that are currently associated with the use of daunorubicin in cancer therapies.

  19. Harmine induces cell cycle arrest and mitochondrial pathway-mediated cellular apoptosis in SW620 cells via inhibition of the Akt and ERK signaling pathways.

    PubMed

    Liu, Jiming; Li, Qiang; Liu, Zhilong; Lin, Liuming; Zhang, Xiangqiang; Cao, Mingrong; Jiang, Jianwei

    2016-06-01

    Harmine, a β-carboline alkaloid isolated from the seeds of Peganum harmala, possesses both antitumor and anti‑nociceptive effects and inhibits human DNA topoisomerase. However, no detailed data are available concerning the mechanisms of harmine in human colorectal carcinoma SW620 cells. In the present study, we demonstrated that harmine inhibited the proliferation of SW620 cells in a dose-dependent manner using MTT and clone formation assays, and the IC50 value of harmine on the growth inhibition of SW620 cells for 48 h was 5.13 µg/ml. PI staining showed that harmine altered the cell cycle distribution by decreasing the proportion of cells in the G0-G1 phase and increasing the proportion in the S and G2-M phase. The expression level of cyclin D1 was decreased, while the expression of cyclin A, E2 and B1, CDK1/cdc2, Myt-1 and p-cdc2 (Tyr15) were increased, which was in accordance with the S and G2/M phase arrest. Hoechst 33258 staining revealed nuclear fragmentation, chromosomal condensation and cell shrinkage in the SW620 cells treated with harmine. Flow cytometry revealed that the percentage of apoptotic sub-G1 cells increased from 7.19 to 26.58%, while in the control group, sub-G1 cells only increased from 1.53 to 1.60%. Furthermore, early and late apoptotic cells were increased from 11.96 to 26.38% when incubated with the indicated concentration of harmine for 48 h, while in the control group, <8% of cells underwent apoptosis. JC-1 staining revealed that harmine decreased mitochondrial transmembrane potential (ΔΨm). The apoptosis of SW620 cells was also detected by western blot analysis, showing caspase-3 and -9, and PARP activation; the downregulation of Bcl-2, Mcl-1, Bcl-xL; and the upregulation of Bax. The expression of p-ERK, p-Akt (Ser473) and p-Akt (Thr308) was inhibited, and phosphorylation of downstream targets of Akt, such as p-FoxO3a and p-GSK‑3β were also attenuated. In conclusion, harmine induces cell cycle arrest and

  20. Preimplantation factor is an anti-apoptotic effector in human trophoblasts involving p53 signaling pathway

    PubMed Central

    Moindjie, Hadia; Santos, Esther Dos; Gouesse, Rita-Josiane; Swierkowski-Blanchard, Nelly; Serazin, Valérie; Barnea, Eytan R; Vialard, François; Dieudonné, Marie-Noëlle

    2016-01-01

    From the earliest stages of gestation, embryonic–maternal interaction has a key role in a successful pregnancy. Various factors present during gestation may significantly influence this type of juxta/paracrine interaction. PreImplantation Factor (PIF) is a recently identified factor with activity at the fetomaternal interface. PIF is secreted by viable embryos and directly controls placental development by increasing the invasive capacity of human extravillous trophoblasts (EVTs). To further specify PIF's role in the human placenta, we analyzed the genome-wide expression profile of the EVT in the presence of a synthetic PIF analog (sPIF). We found that sPIF exposure altered several pathways related to p53 signaling, survival and the immune response. Functional assays revealed that sPIF acts through the p53 pathway to reduce both early and late trophoblast apoptosis. More precisely, sPIF (i) decreases the phosphorylation of p53 at Ser-15, (ii) enhances the B-cell lymphoma-2 (BCL2) expression and (iii) reduces the BCL2-associated X protein (BAX) and BCL2 homologous antagonist killer (BAK) mRNA expression levels. Furthermore, invalidation experiments of TP53 allowed us to demonstrate that PIF's effects on placental apoptosis seemed to be essentially mediated by this gene. We have clearly shown that p53 and sPIF pathways could interact in human trophoblast and thus promotes cell survival. Furthermore, sPIF was found to regulate a gene network related to immune tolerance in the EVT, which emphasizes the beneficial effect of this peptide on the human placenta. Finally, the PIF protein levels in placentas from pregnancies affected by preeclampsia or intra-uterine growth restriction were significantly lower than in gestational age-matched control placentas. Taken as a whole, our results suggest that sPIF protects the EVT's functional status through a variety of mechanisms. Clinical application of sPIF in the treatment of disorders of early pregnancy can be envisioned

  1. Cambogin exerts anti-proliferative and pro-apoptotic effects on breast adenocarcinoma through the induction of NADPH oxidase 1 and the alteration of mitochondrial morphology and dynamics

    PubMed Central

    Xie, Jianling; Wu, Minfeng; Cai, Bo; Liu, Yurong; Zhang, Hong; Tan, Hongsheng; Pan, Yingyi; Xu, Hongxi

    2016-01-01

    Cambogin, a bioactive polycyclic polyprenylated acylphoroglucinol (PPAP) derived from the Garcinia genus, possesses proapoptotic effect in medulloblastoma and breast cancer cells. We have previously demonstrated that the proapoptotic effect of cambogin is driven by the production of reactive oxygen species (ROS). Here we have shown that the inhibitory effect of cambogin on cell proliferation is associated with the loss of mitochondrial transmembrane potential (ΔΨm) and mitochondrial fragmentation. Cambogin also promotes the mutual complex formation of the membrane-bound subunit p22phox of NADPH oxidase 1 (NOX1), as well as the phosphorylation of the cytosolic subunit p47phox, subsequently enhancing membrane-bound NOX1 activity, which leads to increases in intracellular and mitochondrial levels of O2.- and H2O2. Pharmacological inhibition of NOX1 using apocynin (pan-NOX inhibitor), ML171 (NOX1 inhibitor) or siRNA against NOX1 prevents the increases in O2.- and H2O2 levels and the anti-proliferative effect of cambogin. Antioxidants, including SOD (superoxide dismutase), CAT (catalase) and EUK-8, are also able to restore cell viability in the presence of cambogin. Besides, cambogin increases the dissociation of thioredoxin-1 (Trx1) from ASK1, switching the inactive form of ASK1 to the active kinase, subsequently leads to the phosphorylation of JNK/SAPK, which is abolished upon ML171 treatment. The proapoptotic effect of cambogin in breast cancer cells is also aggravated upon knocking down Trx1 in MCF-7 cells. Taken in conjunction, these data indicate that the anti-proliferative and pro-apoptotic effect of cambogin is mediated via inducing NOX1-dependent ROS production and the dissociation of ASK1 and Trx1. PMID:27418140

  2. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

    PubMed

    Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

    2015-01-01

    The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  3. Differential contribution of the mitochondrial translation pathway to the survival of diffuse large B-cell lymphoma subsets.

    PubMed

    Norberg, Erik; Lako, Ana; Chen, Pei-Hsuan; Stanley, Illana A; Zhou, Feng; Ficarro, Scott B; Chapuy, Bjoern; Chen, Linfeng; Rodig, Scott; Shin, Donghyuk; Choi, Dong Wook; Lee, Sangho; Shipp, Margaret A; Marto, Jarrod A; Danial, Nika N

    2017-02-01

    Diffuse large B-cell lymphomas (DLBCLs) are a highly heterogeneous group of tumors in which subsets share molecular features revealed by gene expression profiles and metabolic fingerprints. While B-cell receptor (BCR)-dependent DLBCLs are glycolytic, OxPhos-DLBCLs rely on mitochondrial energy transduction and nutrient utilization pathways that provide pro-survival benefits independent of BCR signaling. Integral to these metabolic distinctions is elevated mitochondrial electron transport chain (ETC) activity in OxPhos-DLBCLs compared with BCR-DLBCLs, which is linked to greater protein abundance of ETC components. To gain insights into molecular determinants of the selective increase in ETC activity and dependence on mitochondrial energy metabolism in OxPhos-DLBCLs, we examined the mitochondrial translation pathway in charge of the synthesis of mitochondrial DNA encoded ETC subunits. Quantitative mass spectrometry identified increased expression of mitochondrial translation factors in OxPhos-DLBCL as compared with the BCR subtype. Biochemical and functional assays indicate that the mitochondrial translation pathway is required for increased ETC activity and mitochondrial energy reserves in OxPhos-DLBCL. Importantly, molecular depletion of several mitochondrial translation proteins using RNA interference or pharmacological perturbation of the mitochondrial translation pathway with the FDA-approved inhibitor tigecycline (Tigecyl) is selectively toxic to OxPhos-DLBCL cell lines and primary tumors. These findings provide additional molecular insights into the metabolic characteristics of OxPhos-DLBCLs, and mark the mitochondrial translation pathway as a potential therapeutic target in these tumors.

  4. The possible FAT1-mediated apoptotic pathways in porcine cumulus cells.

    PubMed

    Wu, Xinhui; Fu, Yao; Sun, Xulei; Liu, Chang; Chai, Menglong; Chen, Chengzhen; Dai, Lisheng; Gao, Yan; Jiang, Hao; Zhang, Jiabao

    2017-01-01

    Porcine cumulus cells are localized around oocytes and act as a specific type of granulosa that plays essential roles in the development and maturation of oocytes, the development and atresia of follicles, and the development of embryos. Studies of FAT1 have demonstrated its functions in cell-cell contact, actin dynamics, and cell growth suppression. To understand whether the FAT1 gene affects the apoptosis of porcine cumulus cells and to elucidate the mechanism of this potential action, FAT1 was knocked down using RNA interference. The lack of FAT1 resulted in stable expression of CTNNB, enhanced expression of cleaved CASP3, but decreased the BCL2/BAX ratios at both the mRNA and protein levels. These results indicated that FAT1 inhibited porcine cumulus cell apoptosis via different pathways. Taken together, these data provide new insights into the mechanisms of the association between FAT1 and porcine cumulus cell apoptosis.

  5. Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody

    PubMed Central

    Thammasit, Patcharin; Sangboonruang, Sirikwan; Suwanpairoj, Supattara; Khamaikawin, Wannisa; Intasai, Nutjeera; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Tragoolpua, Khajornsak

    2015-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer. PMID:25663946

  6. BMSC paracrine activity attenuates interleukin-1β-induced inflammation and apoptosis in rat AF cells via inhibiting relative NF-κB signaling and the mitochondrial pathway

    PubMed Central

    Hu, Jinquan; Yan, Qiang; Shi, Changgui; Tian, Ye; Cao, Peng; Yuan, Wen

    2017-01-01

    We previously showed that bone mesenchymal stem cells (BMSCs) inhibit interleukin-1 beta (IL-1β) induced degenerative effects in NP cells by their paracrine activity, but the anti-inflammatory and anti-apoptotic effect of BMSC paracrine activity and the relative signaling pathway were not further investigated in annulus fibrosus (AF) cells. In this study, AF cells were exposed to IL-1β, which was applied to mimic intervertebral disc degeneration (IDD) in vitro. Indirect co-culture with BMSCs in a transwell co-culture system reduced the activity of nuclear factor-κB-p65 (NF-κB-p65) through the restoration of its inhibitor IκBa. Real time polymerase chain reaction (PT-PCR) and Western blotting revealed that the up-regulation of MMP-3 and MMP-13 induced by IL-1β were impeded by BMSC co-culture, and the decrease in aggrecan, collagen I and TIMP-1 were reversed. An ELISA showed that the increased inflammatory factors, such as nitrite, prostaglandin E-2 (PGE-2), IL-6 and cyclooxygenase-2 (COX-2), were decreased by the BMSC co-culture. Furthermore, the apoptosis rate of AF cells were detected by flow cytometry, and the apoptosis-related proteins, such as Bax, Bcl-2 and caspase-3, were analyzed by Western blotting and ELISA. The changes in mitochondrial membrane potentials were also detected by confocal microscopy. The results showed that IL-1β induced apoptosis of AF cells was attenuated by co-culturing, which suppressed the functions of the mitochondria function. We suggest that BMSC paracrine activity has an anti-inflammation effect and anti-apoptotic effect on IDD, and it is mediated, at least in part, via the relative NF-κF and mitochondrial apoptotic pathways in AF cells. PMID:28123635

  7. The anti-apoptotic form of tyrosine kinase Lyn that is generated by proteolysis is degraded by the N-end rule pathway

    PubMed Central

    Eldeeb, Mohamed A.

    2014-01-01

    The activation of apoptotic pathways results in the caspase cleavage of the Lyn tyrosine kinase to generate the N-terminal truncated LynΔN. This LynΔN fragment has been demonstrated to exert negative feedback on imatinib induced apoptosis in chronic myelogenous leukemia (CML) K562 cells. Our investigations focus on LynΔN stability and how reduced stability reduces imatinib resistance. As the proteolytical generated LynΔN has a leucine as an N-terminal amino acid, we hypothesized that LynΔN would be degraded by the N-end rule pathway. We demonstrated that LynΔN is unstable and that its stability is dependent on the identity of its N-terminus. Additionally we established that LynΔN degradation could be inhibited by either inhibiting the proteasome or knocking down the UBR1 and UBR2 ubiquitin E3 ligases. Importantly, we also demonstrate that LynΔN degradation by the N-end rule counters the imatinib resistance of K562 cells provided by LynΔN expression. Together our data suggest a possible mechanism for the N-end rule pathway having a link to imatinib resistance in CML. With LynΔN being an N-end rule substrate, it provides the first example that this pathway can also provide a pro-apoptotic function as previous reports have currently only demonstrated anti-apoptotic roles for the N-end rule pathway. PMID:24798867

  8. Increased longevity of some C. elegans mitochondrial mutants explained by activation of an alternative energy-producing pathway.

    PubMed

    Gallo, Marco; Park, Donha; Riddle, Donald L

    2011-10-01

    The Caenorhabditis elegans misc-1 gene encodes a mitochondrial carrier with a role in oxidative stress response. The knock-out mutant has no lifespan phenotype and fails to upregulate the gei-7-mediated glyoxylate shunt, an extra-mitochondrial pathway of energy production. We show that gei-7 is required for the longevity of the mitochondrial mutant clk-1. Our data suggest that only mitochondrial mutants that upregulate gei-7 can achieve longevity.

  9. 17β-estradiol impedes Bax-involved mitochondrial apoptosis of retinal nerve cells induced by oxidative damage via the phosphatidylinositol 3-kinase/Akt signal pathway.

    PubMed

    Li, Hongbo; Wang, Baoying; Zhu, Chunhui; Feng, Yan; Wang, Shaolan; Shahzad, Muhammad; Hu, Chenghu; Mo, Mingshu; Du, Fangying; Yu, Xiaorui

    2013-07-01

    Oxidative stress leading to retinal nerve cells (RNCs) apoptosis is a major cause of neurodegenerative disorders of the retina. 17β-Estradiol (E2) has been suggested to be a neuroprotective agent in the central nervous system; however, at present, the underlying mechanisms are not well understood, and the related research on the RNCs is less reported. Here, in order to investigate the protective role and mechanism of E2 against oxidative stress-induced damage on RNCs, the transmission electron microscopy and annexin V-FITC/propidium iodide assay were applied to detect the RNCs apoptosis. Western blot and real-time PCR were used to determine the expression of the critical molecules in Bcl-2 and caspase family associated with apoptosis. The transmission electron microscopy results showed that H(2)O(2) could induce typical features of apoptosis in RNCs, including formation of the apoptosome. E2 could, however, suppress the H(2)O(2)-induced morphological changes of apoptosis. Intriguingly, we observed E2-mediated phagocytic scavenging of apoptosome. In response to H(2)O(2)-induced apoptosis, Bax, acting as one of the pivotal pro-apoptotic members of Bcl-2 family, increased significantly, which directly resulted in an increased ratio of Bax to anti-apoptotic protein Bcl-2 (Bax/Bcl-2). Additionally, caspases 9 and 3, which are the critical molecules of the mitochondrial apoptosis pathway, were activated by H(2)O(2). In contrast, E2 exerted anti-apoptotic effects by reducing the expression of Bax to decrease the ratio of Bax/Bcl-2 and impeded the caspases 9/3 activation. Moreover, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, could sharply block the effect of E2 in reducing the percentage of apoptotic cells resistance to H(2)O(2). And the attenuation of Bax, the reduced activities of caspases 9/3 and the impeded release of mitochondrial cytochrome c mediated by E2 resistance to H(2)O(2) damage were significantly retrieved by LY294002 administration. Taken

  10. Melittin induces human gastric cancer cell apoptosis via activation of mitochondrial pathway

    PubMed Central

    Kong, Gui-Mei; Tao, Wen-Hua; Diao, Ya-Li; Fang, Peng-Hua; Wang, Ji-Jun; Bo, Ping; Qian, Feng

    2016-01-01

    AIM: To investigate the apoptotic effects of melittin on SGC-7901 cells via activation of the mitochondrial signaling pathway in vitro. METHODS: SGC-7901 cells were stimulated by melittin, and its effect on proliferation and apoptosis of was investigated by methyl thiazolyl tetrazolium assay, morphologic structure with transmission electron microscopy, annexin-V/propidium iodide double-staining assay, measuring mitochondrial membrane potential (MMP) levels, and analyzing reactive oxygen species (ROS) concentrations were analyzed by flow cytometry. Cytochrome C (Cyt C), apoptosis-inducing factor (AIF), endonuclease G (Endo G), second mitochondria-derived activator of caspases (Smac)/direct IAP binding protein with low isoelectric point (Diablo), and FAS were analyzed by western blot. The expression of caspase-3 and caspase-8 was measured using activity assay kits. RESULTS: Melittin was incubated at 1.0, 2.0, 4.0, or 6.0 μg/mL for 1, 2, 4, 6, or 8 h and showed a time- and concentration-dependent inhibition of SGC-7901 cell growth. Melittin induced SGC-7901 cell apoptosis, which was confirmed by typical morphological changes. Treatment with 4 μg/mL melittin induced early apoptosis of SGC-7901 cells, and the early apoptosis rates were 39.97% ± 3.19%, 59.27% ± 3.94%, and 71.50% ± 2.87% vs 32.63% ± 2.75% for 1, 2, and 4 h vs 0 h (n = 3, P < 0.05); the ROS levels were 616.53% ± 79.78%, 974.81% ± 102.40%, and 1330.94% ± 93.09% vs 603.74% ± 71.99% (n = 3, P < 0.05); the MMP values were 2.07 ± 0.05, 1.78 ± 0.29, and 1.16 ± 0.25 vs 2.55 ± 0.42 (n = 3, P < 0.05); caspase-3 activity was significantly higher compared to the control (5492.3 ± 321.1, 6562.0 ± 381.3, and 8695.7 ± 449.1 vs 2330.0 ± 121.9), but the caspase activity of the non-tumor cell line L-O2 was not different from that of the control. With the addition of the caspase-3 inhibitor (Ac-DEVD-CHO), caspase-3 activity was significantly decreased compared to the control group (1067.0 ± 132.5 U/g vs

  11. Resveratrol attenuates acute kidney injury by inhibiting death receptor-mediated apoptotic pathways in a cisplatin-induced rat model

    PubMed Central

    Hao, Qiufa; Xiao, Xiaoyan; Zhen, Junhui; Feng, Jinbo; Song, Chun; Jiang, Bei; Hu, Zhao

    2016-01-01

    Acute kidney injury is a clinical syndrome characterized by a loss of renal function and acute tubular necrosis. Resveratrol exerts a wide range of pharmacological effects based on its anti-inflammatory, antioxidant and cytoprotective properties. The present study aimed to evaluate whether resveratrol attenuates acute kidney injury in a cisplatin-induced rat model and to investigate the potential mechanisms involved. Rats were randomly divided into four treatment groups: Control, cisplatin, resveratrol, and cisplatin plus resveratrol. Rats exposed to cisplatin displayed acute kidney injury, identified by analysis of renal function and histopathological observation. Resveratrol significantly ameliorated the increased serum creatinine, blood urea nitrogen, renal index and histopathological damage induced by cisplatin. Furthermore, compared with untreated control animals, cisplatin lead to significantly increased expression of Fas ligand, tumor necrosis factor-α (TNF-α), caspase-8 and Bcl-2 associated protein X apoptosis regulator (Bax), and decreased expression of anti-apoptosis regulators, BH3 interacting domain death agonist (BID) and B cell lymphoma 2 apoptosis regulator (Bcl-2). Administration of resveratrol significantly reversed the cisplatin-induced alteration in these apoptosis-associated proteins. In conclusion, these findings suggest that resveratrol attenuates cisplatin-induced acute kidney injury through inactivation of the death receptor-mediated apoptotic pathway, and may provide a new therapeutic strategy to ameliorate the process of acute kidney injury. PMID:27600998

  12. Apoptotic effect of gambogic acid in esophageal squamous cell carcinoma cells via suppression of the NF-κB pathway

    PubMed Central

    LIU, WEN-YUE; WU, XU; LIAO, CHENG-QUAN; SHEN, JIE; LI, JUN

    2016-01-01

    Despite extensive investigations of therapeutic improvements for surgical techniques, chemotherapy and chemoradiotherapy, esophageal squamous cell carcinoma (ESCC) remains one of the most aggressive forms of cancer, and the prognosis for patients with advanced ESCC remains poor. Therefore, effective therapies are urgently required in order to improve the prognosis of patients with ESCC. TE-1 cells were treated with gambogic acid (GA), and then subjected to western blot analysis, TUNEL assay and caspase activity analysis. GA significantly induced apoptosis in ESCC TE-1 cells. In addition, the antitumor activity of GA was accompanied by the decreased expression of phosphorylated-protein kinase B (p-AKT) and nuclear factor of κ light polypeptide gene enhancer in B-cells 1 (NF-κB). The inhibition of protein kinase B (AKT) and NF-κB activation by chemical inhibitors augmented the apoptotic effect responses to GA in the TE-1 cells. The pan-caspase inhibitor z-VAD-fmk (zVAD) decreased GA-induced apoptosis. Furthermore, zVAD attenuated GA-induced growth inhibition in TE-1 cells. GA induced apoptosis in ESCC TE-1 via suppression of NF-κB pathway. The findings of the present study may provide a novel insight into ESCC treatment. PMID:27284372

  13. Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries.

    PubMed

    Kawasaki, Hiroaki; Onuki, Reiko; Suyama, Eigo; Taira, Kazunari

    2002-04-01

    Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-alpha (TNF-alpha)-mediated pathway of apoptosis, using RNA helicase-associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-alpha-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-alpha-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.

  14. Methyl antcinate A from Antrodia camphorata induces apoptosis in human liver cancer cells through oxidant-mediated cofilin- and Bax-triggered mitochondrial pathway.

    PubMed

    Hsieh, Yun-Chih; Rao, Yerra Koteswara; Wu, Chun-Chi; Huang, Chi-Ying F; Geethangili, Madamanchi; Hsu, Shih-Lan; Tzeng, Yew-Min

    2010-07-19

    We investigated the effects of antcin A, antcin C, and methyl antcinate A (MAA) isolated from Antrodia camphorata on the proliferation of human liver cancer cell lines Huh7, HepG2, and Hep3B and the normal cell rat hepatocytes. The three compounds selectively inhibit the proliferation of tumor cells rather than normal cells, with IC(50) values ranging from 30.2 to 286.4 microM. The compound MAA was a more potent cytotoxic agent than antcins A and C with IC(50) values of 52.2, 78.0, and 30.2 microM against HepG2, Hep3B, and Huh7 cells, respectively. To elucidate the molecular mechanism, treatment of Huh7 cells with 100 microM MAA induced an apoptotic cell death, which was characterized by the appearance of sub-G1 population, DNA fragmentation, TUNEL positive cells, and caspase activation. MAA triggered the mitochondrial apoptotic pathway, as indicated by an increase in the protein expression of Bax, Bak, and PUMA, as well as a decrease in Bcl-(XL) and Bcl-2 and disruption of mitochondrial membrane potential and promotion of mitochondrial cytochrome c release, as well as activation of caspases-2, -3, and -9. We also found that pretreatment with inhibitors of caspases-2, -3, and -9 noticeably blocked MAA-triggered apoptosis. Furthermore, intracellular reactive oxygen species (ROS) generation and NADPH oxidase activation were observed in MAA-stimulated Huh7 cells. Mechanistic studies showed that MAA induces mitochondrial translocation of cofilin. When Huh7 cells were treated with cyclosporine A and bongkrekic acid, an inhibitor of the mitochondria permeability transition pore, the levels of cell death induced by MAA were significantly attenuated. Additionally, pretreatment of Huh7 cells with antioxidants ascorbic acid and N-acetyl cysteine markedly attenuated the MAA-induced apoptosis by upregulation of Bax, Bak, and PUMA, mitochondrial translocation of cofilin, activation of caspase-3, and cell death. Taken together, our results provide the first evidence of the

  15. Synthesis, characterization and apoptotic activity of quinazolinone Schiff base derivatives toward MCF-7 cells via intrinsic and extrinsic apoptosis pathways

    PubMed Central

    Zahedifard, Maryam; Lafta Faraj, Fadhil; Paydar, Mohammadjavad; Yeng Looi, Chung; Hajrezaei, Maryam; Hasanpourghadi, Mohadeseh; Kamalidehghan, Behnam; Abdul Majid, Nazia; Mohd Ali, Hapipah; Ameen Abdulla, Mahmood

    2015-01-01

    The current study investigated the cytotoxic effect of 3-(5-chloro-2-hydroxybenzylideneamino)-2-(5-chloro-2-hydroxyphenyl)-2,3-dihydroquinazolin-41(H)-one (A) and 3-(5-nitro-2-hydroxybenzylideneamino)-2-(5-nitro-2-hydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (B) on MCF-7, MDA-MB-231, MCF-10A and WRL-68 cells. The mechanism involved in apoptosis was assessed to evaluate the possible pathways induced by compound A and B. MTT assay results using A and B showed significant inhibition of MCF-7 cell viability, with IC50 values of 3. 27 ± 0.171 and 4.36 ± 0.219 μg/mL, respectively, after a 72 hour treatment period. Compound A and B did not demonstrate significant cytotoxic effects towards MDA-MB-231, WRL-68 and MCF-10A cells. Acute toxicity tests also revealed an absence of toxic effects on mice. Fluorescent microscopic studies confirmed distinct morphological changes (membrane blebbing and chromosome condensation) corresponding to typical apoptotic features in treated MCF-7 cells. Using Cellomics High Content Screening (HCS), we found that compound A and B could trigger the release of cytochrome c from mitochondria to the cytosol. The release of cytochrome c activated the expression of caspases-9 and then stimulated downstream executioner caspase-3/7. In addition, caspase-8 showed remarkable activity, followed by inhibition of NF-κB activation in A-and B-treated MCF-7 cells. The results indicated that A and B could induce apoptosis via a mechanism that involves either extrinsic or intrinsic pathways. PMID:26108872

  16. Ophiobolin A Induces Autophagy and Activates the Mitochondrial Pathway of Apoptosis in Human Melanoma Cells

    PubMed Central

    Rodolfo, Carlo; Rocco, Mariapina; Cattaneo, Lucia; Tartaglia, Maria; Sassi, Mauro; Aducci, Patrizia; Scaloni, Andrea; Marra, Mauro

    2016-01-01

    Ophiobolin A, a fungal toxin from Bipolaris species known to affect different cellular processes in plants, has recently been shown to have anti-cancer activity in mammalian cells. In the present study, we investigated the anti-proliferative effect of Ophiobolin A on human melanoma A375 and CHL-1 cell lines. This cellular model was chosen because of the incidence of melanoma malignant tumor on human population and its resistance to chemical treatments. Ophyobolin A strongly reduced cell viability of melanoma cells by affecting mitochondrial functionality. The toxin induced depolarization of mitochondrial membrane potential, reactive oxygen species production and mitochondrial network fragmentation, leading to autophagy induction and ultimately resulting in cell death by activation of the mitochondrial pathway of apoptosis. Finally, a comparative proteomic investigation on A375 cells allowed to identify several Ophiobolin A down-regulated proteins, which are involved in fundamental processes for cell homeostasis and viability. PMID:27936075

  17. Apoptotic markers in human blood platelets treated with peroxynitrite.

    PubMed

    Wachowicz, Barbara; Rywaniak, Joanna Zofia; Nowak, Paweł

    2008-12-01

    Platelets are anucleated cells that upon activation by agonists or during storage may develop apoptotic events. The role of peroxynitrite and its reactive intermediates in apoptotic process in blood platelets is unknown. In order to study the appearance of biomarkers of apoptosis in platelets after treatment with peroxynitrite and with thrombin different markers were chosen: annexin V binding (phosphatidylserine exposure), platelet microparticle formation, mitochondrial membrane depolarization, caspase-3 activation and P-selectin expression. In gel-filtrated platelets treated with different concentrations of peroxynitrite (0.01, 0.1, 1.0 mM, 10 minute, 37 degrees C) a significant increase of phosphatidylserine exposure (about 36% at the highest concentration, p < 0.01) and the platelet microparticle formation were observed. Peroxynitrite caused a dose-dependent caspase-3 activation and depolarization of mitochondrial potential. The same apoptotic markers were appeared in thrombin-activated platelets. Dose-dependent tyrosine nitration in platelet proteins caused by peroxynitrite was reduced in the presence of (-)-epicatechin. Moreover, (-)-epicatechin distinctly reduced the level of apoptotic markers. The obtained results indicate that peroxynitrite responsible for oxidative/nitrative stress and changes in platelet function may promote in vitro apoptotic events in human gel-filtrated platelets via intrinsic pathway. Nitration of tyrosine seems to be partly associated with the appearance of apoptotic markers in platelets.

  18. An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation.

    PubMed

    Harner, Max E; Unger, Ann-Katrin; Geerts, Willie Jc; Mari, Muriel; Izawa, Toshiaki; Stenger, Maria; Geimer, Stefan; Reggiori, Fulvio; Westermann, Benedikt; Neupert, Walter

    2016-11-16

    Metabolic function and architecture of mitochondria are intimately linked. More than 60 years ago, cristae were discovered as characteristic elements of mitochondria that harbor the protein complexes of oxidative phosphorylation, but how cristae are formed, remained an open question. Here we present experimental results obtained with yeast that support a novel hypothesis on the existence of two molecular pathways that lead to the generation of lamellar and tubular cristae. Formation of lamellar cristae depends on the mitochondrial fusion machinery through a pathway that is required also for homeostasis of mitochondria and mitochondrial DNA. Tubular cristae are formed via invaginations of the inner boundary membrane by a pathway independent of the fusion machinery. Dimerization of the F1FO-ATP synthase and the presence of the MICOS complex are necessary for both pathways. The proposed hypothesis is suggested to apply also to higher eukaryotes, since the key components are conserved in structure and function throughout evolution.

  19. FoxP3 inhibits proliferation and induces apoptosis of gastric cancer cells by activating the apoptotic signaling pathway

    SciTech Connect

    Ma, Gui-Fen; Chen, Shi-Yao; Sun, Zhi-Rong; Miao, Qing; Liu, Yi-Mei; Zeng, Xiao-Qing; Luo, Tian-Cheng; Ma, Li-Li; Lian, Jing-Jing; Song, Dong-Li

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer The article revealed FoxP3 gene function in gastric cancer firstly. Black-Right-Pointing-Pointer Present the novel roles of FoxP3 in inhibiting proliferation and promoting apoptosis in gastric cancer cells. Black-Right-Pointing-Pointer Overexpression of FoxP3 increased proapoptotic molecules and repressed antiapoptotic molecules. Black-Right-Pointing-Pointer Silencing of FoxP3 reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Black-Right-Pointing-Pointer FoxP3 is sufficient for activating the apoptotic signaling pathway. -- Abstract: Forkhead Box Protein 3 (FoxP3) was identified as a key transcription factor to the occurring and function of the regulatory T cells (Tregs). However, limited evidence indicated its function in tumor cells. To elucidate the precise roles and underlying molecular mechanism of FoxP3 in gastric cancer (GC), we examined the expression of FoxP3 and the consequences of interfering with FoxP3 gene in human GC cell lines, AGS and MKN45, by multiple cellular and molecular approaches, such as immunofluorescence, gene transfection, CCK-8 assay, clone formation assay, TUNEL assay, Flow cytometry, immunoassay and quantities polymerase chain reaction (PCR). As a result, FoxP3 was expressed both in nucleus and cytoplasm of GC cells. Up-regulation of FoxP3 inhibited cell proliferation and promoted cell apoptosis. Overexpression of FoxP3 increased the protein and mRNA levels of proapoptotic molecules, such as poly ADP-ribose polymerase1 (PARP), caspase-3 and caspase-9, and repressed the expression of antiapoptotic molecules, such as cellular inhibitor of apoptosis-1 (c-IAP1) and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2). Furthermore, silencing of FoxP3 by siRNA in GC cells reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Collectively, our findings identify the novel roles of FoxP3 in inhibiting proliferation and inducing apoptosis

  20. How the Wnt signaling pathway protects from neurodegeneration: the mitochondrial scenario

    PubMed Central

    Arrázola, Macarena S.; Silva-Alvarez, Carmen; Inestrosa, Nibaldo C.

    2015-01-01

    Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is characterized by progressive memory loss and cognitive decline. One of the hallmarks of AD is the overproduction of amyloid-beta aggregates that range from the toxic soluble oligomer (Aβo) form to extracellular accumulations in the brain. Growing evidence indicates that mitochondrial dysfunction is a common feature of neurodegenerative diseases and is observed at an early stage in the pathogenesis of AD. Reports indicate that mitochondrial structure and function are affected by Aβo and can trigger neuronal cell death. Mitochondria are highly dynamic organelles, and the balance between their fusion and fission processes is essential for neuronal function. Interestingly, in AD, the process known as “mitochondrial dynamics” is also impaired by Aβo. On the other hand, the activation of the Wnt signaling pathway has an essential role in synaptic maintenance and neuronal functions, and its deregulation has also been implicated in AD. We have demonstrated that canonical Wnt signaling, through the Wnt3a ligand, prevents the permeabilization of mitochondrial membranes through the inhibition of the mitochondrial permeability transition pore (mPTP), induced by Aβo. In addition, we showed that non-canonical Wnt signaling, through the Wnt5a ligand, protects mitochondria from fission-fusion alterations in AD. These results suggest new approaches by which different Wnt signaling pathways protect neurons in AD, and support the idea that mitochondria have become potential therapeutic targets for the treatment of neurodegenerative disorders. Here we discuss the neuroprotective role of the canonical and non-canonical Wnt signaling pathways in AD and their differential modulation of mitochondrial processes, associated with mitochondrial dysfunction and neurodegeneration. PMID:25999816

  1. How the Wnt signaling pathway protects from neurodegeneration: the mitochondrial scenario.

    PubMed

    Arrázola, Macarena S; Silva-Alvarez, Carmen; Inestrosa, Nibaldo C

    2015-01-01

    Alzheimer's disease (AD) is the most common neurodegenerative disorder and is characterized by progressive memory loss and cognitive decline. One of the hallmarks of AD is the overproduction of amyloid-beta aggregates that range from the toxic soluble oligomer (Aβo) form to extracellular accumulations in the brain. Growing evidence indicates that mitochondrial dysfunction is a common feature of neurodegenerative diseases and is observed at an early stage in the pathogenesis of AD. Reports indicate that mitochondrial structure and function are affected by Aβo and can trigger neuronal cell death. Mitochondria are highly dynamic organelles, and the balance between their fusion and fission processes is essential for neuronal function. Interestingly, in AD, the process known as "mitochondrial dynamics" is also impaired by Aβo. On the other hand, the activation of the Wnt signaling pathway has an essential role in synaptic maintenance and neuronal functions, and its deregulation has also been implicated in AD. We have demonstrated that canonical Wnt signaling, through the Wnt3a ligand, prevents the permeabilization of mitochondrial membranes through the inhibition of the mitochondrial permeability transition pore (mPTP), induced by Aβo. In addition, we showed that non-canonical Wnt signaling, through the Wnt5a ligand, protects mitochondria from fission-fusion alterations in AD. These results suggest new approaches by which different Wnt signaling pathways protect neurons in AD, and support the idea that mitochondria have become potential therapeutic targets for the treatment of neurodegenerative disorders. Here we discuss the neuroprotective role of the canonical and non-canonical Wnt signaling pathways in AD and their differential modulation of mitochondrial processes, associated with mitochondrial dysfunction and neurodegeneration.

  2. A Rab5 endosomal pathway mediates Parkin-dependent mitochondrial clearance.

    PubMed

    Hammerling, Babette C; Najor, Rita H; Cortez, Melissa Q; Shires, Sarah E; Leon, Leonardo J; Gonzalez, Eileen R; Boassa, Daniela; Phan, Sébastien; Thor, Andrea; Jimenez, Rebecca E; Li, Hong; Kitsis, Richard N; Dorn Ii, Gerald W; Sadoshima, Junichi; Ellisman, Mark H; Gustafsson, Åsa B

    2017-01-30

    Damaged mitochondria pose a lethal threat to cells that necessitates their prompt removal. The currently recognized mechanism for disposal of mitochondria is autophagy, where damaged organelles are marked for disposal via ubiquitylation by Parkin. Here we report a novel pathway for mitochondrial elimination, in which these organelles undergo Parkin-dependent sequestration into Rab5-positive early endosomes via the ESCRT machinery. Following maturation, these endosomes deliver mitochondria to lysosomes for degradation. Although this endosomal pathway is activated by stressors that also activate mitochondrial autophagy, endosomal-mediated mitochondrial clearance is initiated before autophagy. The autophagy protein Beclin1 regulates activation of Rab5 and endosomal-mediated degradation of mitochondria, suggesting cross-talk between these two pathways. Abrogation of Rab5 function and the endosomal pathway results in the accumulation of stressed mitochondria and increases susceptibility to cell death in embryonic fibroblasts and cardiac myocytes. These data reveal a new mechanism for mitochondrial quality control mediated by Rab5 and early endosomes.

  3. A Rab5 endosomal pathway mediates Parkin-dependent mitochondrial clearance

    PubMed Central

    Hammerling, Babette C.; Najor, Rita H.; Cortez, Melissa Q.; Shires, Sarah E.; Leon, Leonardo J.; Gonzalez, Eileen R.; Boassa, Daniela; Phan, Sébastien; Thor, Andrea; Jimenez, Rebecca E.; Li, Hong; Kitsis, Richard N.; Dorn II, Gerald W.; Sadoshima, Junichi; Ellisman, Mark H.; Gustafsson, Åsa B.

    2017-01-01

    Damaged mitochondria pose a lethal threat to cells that necessitates their prompt removal. The currently recognized mechanism for disposal of mitochondria is autophagy, where damaged organelles are marked for disposal via ubiquitylation by Parkin. Here we report a novel pathway for mitochondrial elimination, in which these organelles undergo Parkin-dependent sequestration into Rab5-positive early endosomes via the ESCRT machinery. Following maturation, these endosomes deliver mitochondria to lysosomes for degradation. Although this endosomal pathway is activated by stressors that also activate mitochondrial autophagy, endosomal-mediated mitochondrial clearance is initiated before autophagy. The autophagy protein Beclin1 regulates activation of Rab5 and endosomal-mediated degradation of mitochondria, suggesting cross-talk between these two pathways. Abrogation of Rab5 function and the endosomal pathway results in the accumulation of stressed mitochondria and increases susceptibility to cell death in embryonic fibroblasts and cardiac myocytes. These data reveal a new mechanism for mitochondrial quality control mediated by Rab5 and early endosomes. PMID:28134239

  4. Alkyl Cinnamates Induce Protein Kinase C Translocation and Anticancer Activity against Breast Cancer Cells through Induction of the Mitochondrial Pathway of Apoptosis

    PubMed Central

    Deka, Suman Jyoti; Mamdi, Narsimha; Manna, Debasis

    2016-01-01

    Purpose The protein kinase C (PKC) family of serine-threonine kinases plays an important role in cancer cell progression. Thus, molecules that target PKC have potential as anticancer agents. The current study aims to understand the treatment of breast cancer cells with alkyl cinnamates. We have also explored the mechanistic details of their anticancer action and the underlying molecular signaling. Methods 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure the viability of MDAMB-231 breast cancer cells to assess the anticancer activity of these compounds. In addition, flow cytometry was performed to study the effect of alkyl cinnamates on the cell cycle and apoptosis. Immunoblotting and immunofluorescence techniques were performed to study PKC translocation, cytochrome c release, and modulation of the mitochondrial membrane potential in breast cancer cells targeted with alkyl cinnamates. Results The PKC agonist DM-2-8 translocated 16.6%±1.7% PKCα from cytosol to the plasma membrane and showed excellent anticancer activity with an half maximal inhibitory concentration (IC50) of 4.13±0.27 µg/mL against cancer cells. The treated cells had an abnormal morphology and exhibited cell cycle defects with G2/M arrest and reduced S phase. Cancer cells treated with DM-2-3, DM-2-4, or DM-2-8 underwent apoptosis as the major pathway of cell death, further confirmed by genomic DNA fragmentation. Furthermore, the mitochondrial membrane potential was perturbed, indicating involvement of the mitochondrial pathway of apoptosis. Immunolocalization studies revealed cytochrome c release from mitochondria to cytosol. Cancer cells treated with DM-2-8 and curcumin showed activation of caspase-9 and caspase-3 as downstream molecular components of the apoptotic pathway. Alkyl cinnamates also caused oxidative stress, which regulates the apoptotic machinery (DNA fragmentation), cell death, and morphological abnormalities in cancer cells

  5. The Infertility of Repeat-Breeder Cows During Summer Is Associated with Decreased Mitochondrial DNA and Increased Expression of Mitochondrial and Apoptotic Genes in Oocytes.

    PubMed

    Ferreira, Roberta Machado; Chiaratti, Marcos Roberto; Macabelli, Carolina Habermann; Rodrigues, Carlos Alberto; Ferraz, Márcio Leão; Watanabe, Yeda Fumie; Smith, Lawrence Charles; Meirelles, Flávio Vieira; Baruselli, Pietro Sampaio

    2016-03-01

    Oocyte quality is known to be a major cause of infertility in repeat-breeder (RB) and heat-stressed dairy cows. However, the mechanisms by which RB oocytes become less capable of supporting embryo development remain largely unknown. Thus, the aim of this study was to investigate whether the decreased oocyte competence of RB cows (RBs) during summer is associated with an altered gene expression profile and a decrease in mitochondrial DNA (mtDNA) copy number. Therefore, oocytes collected from heifers, non-RBs in peak lactation (PLs), and RBs were used to evaluate mtDNA amounts as well as the expression levels of genes associated with the mitochondria (MT-CO1, NRF1, POLG, POLG2, PPARGC1A, and TFAM), apoptosis (BAX, BCL2, and ITM2B), and oocyte maturation (BMP15, FGF8, FGF10, FGF16, FGF17, and GDF9). The oocytes retrieved from RBs during winter contained over eight times more mtDNA than those retrieved from RBs during summer. They also contained significantly less mtDNA than oocytes retrieved from heifers and PLs during summer. Moreover, the expression of mitochondria- (NRF1, POLG, POLG2, PPARGC1A, and TFAM) and apoptosis-related (BAX and ITM2B) genes, as well as of GDF9, in RB oocytes collected during summer was significantly greater than that in oocytes collected from heifers and PLs during the same season. In oocytes from heifers and PLs, the expression levels of these genes were lower in those collected during summer compared with winter, but this difference was not observed in oocytes collected from RBs. Altogether, these data provide evidence of altered gene expression and reduced mtDNA copy number in the oocytes collected from RBs during summer. This indicates a loss of fertility in RBs during summer, which might be caused by a possible mitochondrial dysfunction associated with a greater chance of oocytes to undergo apoptosis.

  6. Tl(I) and Tl(III) activate both mitochondrial and extrinsic pathways of apoptosis in rat pheochromocytoma (PC12) cells

    SciTech Connect

    Hanzel, Cecilia Eliana; Verstraeten, Sandra Viviana

    2009-04-01

    Thallium (Tl) is a highly toxic metal though yet its mechanisms are poorly understood. Previously, we demonstrated that rat pheochromocytoma (PC12) cells exposure to thallous (Tl(I)) or thallic (Tl(III)) cations leads to mitochondrial damage and reduced cell viability. In the present work we comparatively characterized the possible pathways involved in Tl(I)- and Tl(III)- (10-100 {mu}M) mediated decrease in PC12 cells viability. We observed that these cations do not cause cell necrosis but significantly increased the number of cells with apoptotic features. Both cations lead to Bax oligomerization and caused apoptosis inducing factor (AIF), endonuclease G (Endo G), and cytochrome c release from mitochondria, but they did not activate caspase dependent DNAse (CAD). Tl(I)- and Tl(III)-dependent caspases 9 and 3 activation followed similar kinetics, with maximal effects at 18 h of incubation. In addition, Tl(I) promoted phosphatidylserine (PS) exposure. Tl(III) induced 2- and 18-fold increase in Fas content and caspase 8 activity, respectively. Together, experimental results show that Tl(I) and Tl(III) induce PC12 cells apoptosis, although differential pathways are involved. While Tl(I)-mediated cell apoptosis was mainly associated with mitochondrial damage, Tl(III) showed a mixed effect triggering both the intrinsic and extrinsic pathways of apoptosis. These findings contribute to a better understanding of the mechanisms underlying Tl-induced loss of cell viability in PC12 cells.

  7. TSPO: functions and applications of a mitochondrial stress response pathway.

    PubMed

    Campanella, Michelangelo; Turkheimer, Federico E

    2015-08-01

    The mitochondrial outer membrane protein TSPO (translocator protein) lies in a privileged position at the interface between mitochondrion and cytosol. Since the initially discovery, nearly forty years ago, it has generated major interest among various disciplines of modern experimental and applied biomedicine. The focused meeting we have organized aimed at summarizing the state of the art knowledge on TSPO and the discipline-based segregated concepts that have made this an exciting and active field of science. The scientists who have generously contributed the event have agreed to generate a special issue here published--stemmed from the discussion of the vent. This consists in a series of contributions via which the know-how is shared aiming to inspire current and future endeavours to validate and accelerate the impact of TSPO science in human pathophysiology and clinical applications.

  8. Data-driven modeling of SRC control on the mitochondrial pathway of apoptosis: implication for anticancer therapy optimization.

    PubMed

    Ballesta, Annabelle; Lopez, Jonathan; Popgeorgiev, Nikolay; Gonzalo, Philippe; Doumic, Marie; Gillet, Germain

    2013-04-01

    Src tyrosine kinases are deregulated in numerous cancers and may favor tumorigenesis and tumor progression. We previously described that Src activation in NIH-3T3 mouse fibroblasts promoted cell resistance to apoptosis. Indeed, Src was found to accelerate the degradation of the pro-apoptotic BH3-only protein Bik and compromised Bax activation as well as subsequent mitochondrial outer membrane permeabilization. The present study undertook a systems biomedicine approach to design optimal anticancer therapeutic strategies using Src-transformed and parental fibroblasts as a biological model. First, a mathematical model of Bik kinetics was designed and fitted to biological data. It guided further experimental investigation that showed that Bik total amount remained constant during staurosporine exposure, and suggested that Bik protein might undergo activation to induce apoptosis. Then, a mathematical model of the mitochondrial pathway of apoptosis was designed and fitted to experimental results. It showed that Src inhibitors could circumvent resistance to apoptosis in Src-transformed cells but gave no specific advantage to parental cells. In addition, it predicted that inhibitors of Bcl-2 antiapoptotic proteins such as ABT-737 should not be used in this biological system in which apoptosis resistance relied on the deficiency of an apoptosis accelerator but not on the overexpression of an apoptosis inhibitor, which was experimentally verified. Finally, we designed theoretically optimal therapeutic strategies using the data-calibrated model. All of them relied on the observed Bax overexpression in Src-transformed cells compared to parental fibroblasts. Indeed, they all involved Bax downregulation such that Bax levels would still be high enough to induce apoptosis in Src-transformed cells but not in parental ones. Efficacy of this counterintuitive therapeutic strategy was further experimentally validated. Thus, the use of Bax inhibitors might be an unexpected way to

  9. Deep hypothermia-enhanced autophagy protects PC12 cells against oxygen glucose deprivation via a mitochondrial pathway.

    PubMed

    Tang, Dang; Wang, Cheng; Gao, Yongjun; Pu, Jun; Long, Jiang; Xu, Wei

    2016-10-06

    Deep hypothermia is known for its organ-preservation properties, which is introduced into surgical operations on the brain and heart, providing both safety in stopping circulation as well as an attractive bloodless operative field. However, the molecular mechanisms have not been clearly identified. This study was undertaken to determine the influence of deep hypothermia on neural apoptosis and the potential mechanism of these effects in PC12 cells following oxygen-glucose deprivation. Deep hypothermia (18°C) was given to PC12 cells while the model of oxygen-glucose deprivation (OGD) induction for 1h. After 24h of reperfusion, the results showed that deep hypothermia decreased the neural apoptosis, and significantly suppressed overexpression of Bax, CytC, Caspase 3, Caspase 9 and cleaved PARP-1, and inhibited the reduction of Bcl-2 expression. While deep hypothermia increased the LC3II/LC3I and Beclin 1, an autophagy marker, which can be inhibited by 3-methyladenine (3-MA), indicating that deep hypothermia-enhanced autophagy ameliorated apoptotic cell death in PC12 cells subjected to OGD. Based on these findings we propose that deep hypothermia protects against neural apoptosis after the induction of OGD by attenuating the mitochondrial apoptosis pathway, moreover, the mechanism of these antiapoptosis effects is related to the enhancement of autophagy, which autophagy might provide a means of neuroprotection against OGD.

  10. Glycycoumarin inhibits hepatocyte lipoapoptosis through activation of autophagy and inhibition of ER stress/GSK-3-mediated mitochondrial pathway

    PubMed Central

    Zhang, Enxiang; Yin, Shutao; Song, Xinhua; Fan, Lihong; Hu, Hongbo

    2016-01-01

    Herbal medicine as an alternative approach in the treatment of disease has drawn growing attention. Identification of the active ingredient is needed for effective utilization of the herbal medicine. Licorice is a popular herbal plant that is widely used to treat various diseases including liver diseases. Glycycoumarin (GCM) is a representative of courmarin compounds isolated from licorice. In the present study, the protective effect of GCM on hepatocyte lipoapoptosis has been evaluated using both cell culture model of palmitate-induced lipoapoptosis and animal model of non-alcoholic steatohepatitis (NASH). The results demonstrated for the first time that GCM was highly effective in suppressing hepatocyte lipoapoptosis in both in vitro and in vivo. Mechanistically, GCM was able to re-activate the impaired autophagy by lipid metabolic disorders. In line with the activation of autophagy, ER stress-mediated JNK and mitochondrial apoptotic pathway activation was inhibited by GCM both in vitro and in vivo. In addition, inactivation of GSK-3 might also contribute to the protective effect of GCM on hepatocyte lipoapoptosis. Our findings supported GCM as a novel active component of licorice against non-alcoholic fatty liver disease (NAFLD). PMID:27901086

  11. Downstream modulation of extrinsic apoptotic pathway in streptozotocin-induced Alzheimer's dementia in rats: Erythropoietin versus curcumin.

    PubMed

    Samy, Doaa M; Ismail, Cherine A; Nassra, Rasha A; Zeitoun, Teshreen M; Nomair, Azhar M

    2016-01-05

    Erythropoietin and curcumin showed promising neuroprotective effects in various models of Alzheimer's dementia. This study was designed to compare the beneficial effects of erythropoietin and/or curcumin in intracerebro-ventricular (ICV) streptozotocin-induced Alzheimer's like disease in rats. Rats received ICV injection of either saline (control, n=8 rats), or streptozotocin. Three weeks following surgery, streptozotocin-injected rats were assigned into 4 groups (8 rats each); vehicle, curcumin (80mg/kg/day, orally), erythropoietin (500 IU/kg every other day, intraperitoneally) and combined (curcumin and erythropoietin)-treated groups. After 3 months of treatment, rats were subjected to neurobehavioral testing, and then killed for biochemical and histological assessment of hippocampus. Fas ligand protein and caspase-8 activity as mediators of extrinsic apoptotic pathway, oxidative stress markers (malondialdehyde and reduced glutathione) and β-amyloid (1-40 and 1-42) peptides were measured. The results showed that administration of erythropoietin suppressed extrinsic apoptosis better than curcumin, while curcumin was more effective in combating oxidative stress in ICV-streptozotocin injected rats. Both erythropoietin and curcumin treatments (individually or combined) equally reduced the hippocampal β-amyloid accumulation and improved cognitive impairment in Morris water maze and passive avoidance tasks. The combined treatment was the most effective in ameliorating apoptosis and oxidative stress rather than behavioral responses or β-amyloid burden. In conclusion, ICV-streptozotocin-induced Alzheimer's dementia activates hippocampal Fas ligand-mediated apoptosis, which could be reduced by erythropoietin and/or curcumin treatment. Curcumin supplementation alone could ameliorate cognitive deficits and reverse biochemical alterations in ICV-streptozotocin Alzheimer's rat model without the hazardous polycythemic effect of long-term erythropoietin injection.

  12. Molecular and Computational Studies on Apoptotic Pathway Regulator, Bcl-2 Gene from Breast Cancer Cell Line MCF-7.

    PubMed

    Tiwari, Pragya; Khan, M J

    2016-01-01

    Cancer is a dreadful disease constituting abnormal growth and proliferation of malignant cells in the body. Next to lung cancer, breast cancer is the most common form of cancer affecting women. The apoptotic pathway regulators, B cell lymphoma family of protein, play a key role in various malignancies defining cancer and their constitutive expression plays an integral role in breast cancer chemotherapy. The research work discusses the identification and molecular cloning of a B cell lymphoma like gene from human breast cancer cell line. The open reading frame of the gene consisted of 965 nucleotides, encoding a protein of 380 amino acids with a predicted molecular weight of 42.5 kilodalton. The predicted physiochemical properties of the gene were as follows: Isoelectric point - 9.49, molecular formula - C1893H3004N534O548S16, total number of negatively charged residues, (Aspartate+Glutamate) - 26, total number of positively charged residues, (Arginine+Lysine)-39, instability index-42.08 (unstable protein) and grand average of hydropathicity is -0.202. Additionally, phobius prediction suggested non-cytoplasmic localization of the putative protein. The presence of secondary structure in the protein was determined by Memsat program. A 3 dimensional protein homology model was generated using threading based method of protein modeling for structural and functional annotation of the putative protein. Future prospects accounts for the biochemical characterization of the enzyme including in vitro assays on breast cancer cell line would establish the functional characteristics of the protein and its physiological mechanisms in breast cancer development and its therapeutic-target role in future.

  13. Differential submitochondrial localization of PINK1 as a molecular switch for mediating distinct mitochondrial signaling pathways.

    PubMed

    Fallaize, Dana; Chin, Lih-Shen; Li, Lian

    2015-12-01

    Mutations in mitochondrial kinase PINK1 cause Parkinson disease (PD), but the submitochondrial site(s) of PINK1 action remains unclear. Here, we report that three-dimensional structured illumination microscopy (3D-SIM) enables super-resolution imaging of protein submitochondrial localization. Dual-color 3D-SIM imaging analysis revealed that PINK1 resides in the cristae membrane and intracristae space but not on the outer mitochondrial membrane (OMM) of healthy mitochondria. Under normal physiological conditions, PINK1 colocalizes with its substrate TRAP1 in the cristae membrane and intracristae space. In response to mitochondrial depolarization, PINK1, but not TRAP1, translocates to the OMM. The PINK1 translocation to the OMM of depolarized mitochondria is independent of new protein synthesis and requires combined action of PINK1 transmembrane domain and C-terminal region. We found that mitochondrial depolarization-induced PINK1 OMM translocation is required for recruitment of parkin to the OMM of damaged mitochondria. Our findings suggest that differential submitochondrial localization of PINK1 serves as a molecular switch for mediating two distinct mitochondrial signaling pathways in maintenance of mitochondrial homeostasis. Furthermore, our study provides evidence for the involvement of deregulated PINK1 submitochondrial localization in PD pathogenesis.

  14. A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death

    PubMed Central

    Wang, Z; Liu, D; Varin, A; Nicolas, V; Courilleau, D; Mateo, P; Caubere, C; Rouet, P; Gomez, A-M; Vandecasteele, G; Fischmeister, R; Brenner, C

    2016-01-01

    Although cardiac cytosolic cyclic 3′,5′-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3−) and Ca2+, sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca2+ entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na+/Ca2+ exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3− rescued the sensitization of mitochondria to Ca2+-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies. PMID:27100892

  15. Non-apoptotic functions of BCL-2 family proteins.

    PubMed

    Gross, Atan; Katz, Samuel G

    2017-02-24

    The BCL-2 family proteins are major regulators of the apoptosis process, but the mechanisms by which they regulate this process are only partially understood. It is now well documented that these proteins play additional non-apoptotic roles that are likely to be related to their apoptotic roles and to provide important clues to cracking their mechanisms of action. It seems that these non-apoptotic roles are largely related to the activation of cellular survival pathways designated to maintain or regain cellular survival, but, if unsuccessful, will switch over into a pro-apoptotic mode. These non-apoptotic roles span a wide range of processes that include the regulation of mitochondrial physiology (metabolism, electron transport chain, morphology, permeability transition), endoplasmic reticulum physiology (calcium homeostasis, unfolded protein response (UPR)), nuclear processes (cell cycle, DNA damage response (DDR)), whole-cell metabolism (glucose and lipid), and autophagy. Here we review all these different non-apoptotic roles, make an attempt to link them to the apoptotic roles, and present many open questions for future research directions in this fascinating field.Cell Death and Differentiation advance online publication, 24 February 2017; doi:10.1038/cdd.2017.22.

  16. Protective role of mitochondrial unsaturated lipids on the preservation of the apoptotic ability of cytochrome C exposed to singlet oxygen.

    PubMed

    Rodrigues, Tiago; de França, Lucimar P; Kawai, Cintia; de Faria, Priscila A; Mugnol, Katia C U; Braga, Fernanda M; Tersariol, Ivarne L S; Smaili, Soraya S; Nantes, Iseli L

    2007-08-31

    Cytochrome c-mediated apoptosis in cells submitted to photodynamic therapy raises the question about the ability of photodynamically oxidized cytochrome c (cytc405) to trigger apoptosis as well as the effect of membranes on protein photo-oxidation. Cytochrome c was submitted to irradiation in the presence of MB+ in phosphate buffer and in the presence of four types of phosphatidylcholine/phosphatidylethanolamine/cardiolipin (PCPECL) liposomes (50/30/20%): totally saturated lipids (tsPCPECL), totally unsaturated lipids (tuPCPECL), partially unsaturated (80%) lipids, with unsaturation in the PC and PE content (puPCPECL80), and partially unsaturated (20%) lipids, with unsaturation in the CL content (puPCPECL20). Cytc405 was formed by irradiation in buffered water and in tsPCPECL and puPCPECL20 liposomes. In the presence of tuPCPECL and puPCPECL80, cytochrome c was protected from photodynamic damage (lipid-protected cytochrome c). In CL liposomes, 25% unsaturated lipids were enough to protect cytochrome c. The presence of unsaturated lipids, in amounts varying according to the liposome composition, are crucial to protect cytochrome c. Interesting findings corroborating the unsaturated lipids as cytochrome c protectors were obtained from the analysis of the lipid-oxidized derivatives of the samples. Native cytochrome c, lipid-protected cytochrome c, and cytc405 were microinjected in aortic smooth muscle cells. Apoptosis, characterized by nucleus blebbing and chromatin condensation, was detected in cells loaded with native and lipid protected cytochrome c but not in cells loaded with cytc405. These results suggest that photodynamic therapy-promoted apoptosis is feasible due to the protective effect of the mitochondrial lipids on the cytochrome c structure and function.

  17. Platinum(IV) complex LA-12 exerts higher ability than cisplatin to enhance TRAIL-induced cancer cell apoptosis via stimulation of mitochondrial pathway.

    PubMed

    Jelínková, Iva; Šafaříková, Barbora; Vondálová Blanářová, Olga; Skender, Belma; Hofmanová, Jiřina; Sova, Petr; Moyer, Mary Pat; Kozubík, Alois; Kolář, Zdeněk; Ehrmann, Jiří; Hyršlová Vaculová, Alena

    2014-12-01

    In search for novel strategies in colon cancer treatment, we investigated the unique ability of platinum(IV) complex LA-12 to efficiently enhance the killing effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), and compared it with the sensitizing action of cisplatin. We provide the first evidence that LA-12 primes human colon cancer cells for TRAIL-induced cytotoxicity by p53-independent activation of the mitochondrial apoptotic pathway. The cooperative action of LA-12 and TRAIL was associated with stimulation of Bax/Bak activation, drop of mitochondrial membrane potential, caspase-9 activation, and a shift of the balance among Bcl-2 family proteins in favor of the pro-apoptotic members. In contrast to cisplatin, LA-12 was a potent inducer of ERK-mediated Noxa and BimL protein upregulation, and more effectively enhanced TRAIL-induced apoptosis in the absence of Bax. The cooperative action of LA-12 and TRAIL was augmented following the siRNA-mediated silencing of Mcl-1 in both Bax proficient/deficient cells. We newly demonstrated that LA-12 induced ERK-mediated c-Myc upregulation, and proved that c-Myc silencing inhibited the mitochondrial activation and apoptosis in colon cancer cells treated with LA-12 and TRAIL. The LA-12-mediated sensitization to TRAIL-induced apoptosis was demonstrated in several colon cancer cell lines, further underscoring the general relevance of our findings. The selective action of LA-12 was documented by preferential priming of cancer but not normal colon cancer cells to TRAIL killing effects. Our work highlights the promising potential of LA-12 over cisplatin to enhance the colon cancer cell sensitivity to TRAIL-induced apoptosis, and provides new mechanistic insights into their cooperative action.

  18. Organization of the human mitochondrial hydrogen sulfide oxidation pathway.

    PubMed

    Libiad, Marouane; Yadav, Pramod Kumar; Vitvitsky, Victor; Martinov, Michael; Banerjee, Ruma

    2014-11-07

    Sulfide oxidation is expected to play an important role in cellular switching between low steady-state intracellular hydrogen sulfide levels and the higher concentrations where the physiological effects are elicited. Yet despite its significance, fundamental questions regarding how the sulfide oxidation pathway is wired remain unanswered, and competing proposals exist that diverge at the very first step catalyzed by sulfide quinone oxidoreductase (SQR). We demonstrate that, in addition to sulfite, glutathione functions as a persulfide acceptor for human SQR and that rhodanese preferentially synthesizes rather than utilizes thiosulfate. The kinetic behavior of these enzymes provides compelling evidence for the flow of sulfide via SQR to glutathione persulfide, which is then partitioned to thiosulfate or sulfite. Kinetic simulations at physiologically relevant metabolite concentrations provide additional support for the organizational logic of the sulfide oxidation pathway in which glutathione persulfide is the first intermediate formed.

  19. Apoptotic cell death in rat epididymis following epichlorohydrin treatment.

    PubMed

    Lee, I-C; Kim, K-H; Kim, S-H; Baek, H-S; Moon, C; Kim, S-H; Yun, W-K; Nam, K-H; Kim, H-C; Kim, J-C

    2013-06-01

    Epichlorohydrin (ECH) is an antifertility agent that acts both as an epididymal toxicant and an agent capable of directly affecting sperm motility. This study identified the time course of apoptotic cell death in rat epididymides after ECH treatment. Rats were administrated with a single oral dose of ECH (50 mg/kg). ECH-induced apoptotic changes were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and its related mechanism was confirmed by Western blot analysis and colorimetric assay. The TUNEL assay showed that the number of apoptotic cells increased at 8 h, reached a maximum level at 12 h, and then decreased progressively. The Western blot analysis demonstrated no significant changes in proapoptotic Bcl-2-associated X (Bax) and anti-apoptotic Bcl-2 expression during the time course of the study. However, phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) and phospho-c-Jun amino-terminal kinase (p-JNK) expression increased at 8-24 h. Caspase-3 and caspase-8 activities also increased at 8-48 h and 12-48 h, respectively, in the same manner as p-p38 MAPK and p-JNK expression. These results indicate that ECH induced apoptotic changes in rat epididymides and that the apoptotic cell death may be related more to the MAPK pathway than to the mitochondrial pathway.

  20. Insulin Stimulates Mitochondrial Fusion and Function in Cardiomyocytes via the Akt-mTOR-NFκB-Opa-1 Signaling Pathway

    PubMed Central

    Parra, Valentina; Verdejo, Hugo E.; Iglewski, Myriam; del Campo, Andrea; Troncoso, Rodrigo; Jones, Deborah; Zhu, Yi; Kuzmicic, Jovan; Pennanen, Christian; Lopez‑Crisosto, Camila; Jaña, Fabián; Ferreira, Jorge; Noguera, Eduard; Chiong, Mario; Bernlohr, David A.; Klip, Amira; Hill, Joseph A.; Rothermel, Beverly A.; Abel, Evan Dale; Zorzano, Antonio; Lavandero, Sergio

    2014-01-01

    Insulin regulates heart metabolism through the regulation of insulin-stimulated glucose uptake. Studies have indicated that insulin can also regulate mitochondrial function. Relevant to this idea, mitochondrial function is impaired in diabetic individuals. Furthermore, the expression of Opa-1 and mitofusins, proteins of the mitochondrial fusion machinery, is dramatically altered in obese and insulin-resistant patients. Given the role of insulin in the control of cardiac energetics, the goal of this study was to investigate whether insulin affects mitochondrial dynamics in cardiomyocytes. Confocal microscopy and the mitochondrial dye MitoTracker Green were used to obtain three-dimensional images of the mitochondrial network in cardiomyocytes and L6 skeletal muscle cells in culture. Three hours of insulin treatment increased Opa-1 protein levels, promoted mitochondrial fusion, increased mitochondrial membrane potential, and elevated both intracellular ATP levels and oxygen consumption in cardiomyocytes in vitro and in vivo. Consequently, the silencing of Opa-1 or Mfn2 prevented all the metabolic effects triggered by insulin. We also provide evidence indicating that insulin increases mitochondrial function in cardiomyocytes through the Akt-mTOR-NFκB signaling pathway. These data demonstrate for the first time in our knowledge that insulin acutely regulates mitochondrial metabolism in cardiomyocytes through a mechanism that depends on increased mitochondrial fusion, Opa-1, and the Akt-mTOR-NFκB pathway. PMID:24009260

  1. Fucoidan inhibits proliferation of the SKM-1 acute myeloid leukaemia cell line via the activation of apoptotic pathways and production of reactive oxygen species.

    PubMed

    Wei, Chunmei; Xiao, Qing; Kuang, Xingyi; Zhang, Tao; Yang, Zesong; Wang, Li

    2015-11-01

    Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid disorders characterized by peripheral blood cytopenias and a high risk of progression to acute myeloid leukaemia (AML). Fucoidan, a complex sulphated polysaccharide isolated from the cell wall of brown seaweeds, has recently attracted attention for its multiple biological activities and its potential as a novel candidate for cancer therapy. In the present study, the anti‑cancer activity of fucoidan was investigated in the MDS/AML cell line SKM‑1. Fucoidan inhibited proliferation, induced apoptosis and caused G1-phase arrest of the cell cycle in SKM‑1 cells as determined by a cell counting kit 8 assay and flow cytometry. Furthermore, reverse transcription quantitative polymerase chain reaction and western blot analyses indicated that treatment with fucoidan (100 µg/ml for 48 h) activated Fas and caspase‑8 in SKM‑1 cells, which are critical for the extrinsic apoptotic pathway; furthermore, caspase‑9 was activated via decreases in phosphoinositide-3 kinase/Akt signaling as indicated by reduced levels of phosphorylated Akt, suggesting the involvement of the intrinsic apoptotic pathway. In addition, fucoidan treatment of SKM‑1 cells resulted in the generation of reactive oxygen species (ROS) as determined by staining with dichloro-dihydro-fluorescein diacetate. These results suggested that the mechanisms of the anti‑cancer effects of fucoidan in SKM‑1 are closely associated with cell cycle arrest and apoptotic cell death, which partly attributed to the activation of apoptotic pathways and accumulation of intracellular ROS. Our results demonstrated that Fucoidan inhibits proliferation and induces the apoptosis of SKM‑1 cells, which provides substantial therapeutic potential for MDS treatment.

  2. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    SciTech Connect

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G.

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  3. The mitochondrial and death receptor pathways involved in the thymocytes apoptosis induced by aflatoxin B1

    PubMed Central

    Chi, Xiaofeng; Li, Xiaochong; Jiang, Min; Fang, Jing; Cui, Hengmin; Lai, Weimin; Zhou, Yi; Zhou, Shan

    2016-01-01

    Aflatoxin B1 (AFB1) is a potent immunosuppressive agent in endotherms, which can be related to the up-regulated apoptosis of immune organs. In this study, we investigated the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in Aflatoxin B1 induced thymocytes apoptosis. Chickens were fed an aflatoxin B1 containing diet (0.6 mg/kg AFB1) for 3 weeks. Our results showed that (1) AFB1 diet induced the decrease of T-cell subsets, morphological changes, and excessive apoptosis of thymus. (2) The excessive apoptosis involved the mitochondrial pathway (up-regulation of Bax, Bak, cytC and down-regulation of Bcl-2 and Bcl-xL) and death receptor pathway (up-regulation of FasL, Fas and FADD). (3) Oxidative stress, an apoptosis inducer, was confirmed in the thymus. In conclusion, this is the first study to demonstrate that mitochondrial and death receptor pathways involved in AFB1 induced thymocytes apoptosis in broilers. PMID:26933817

  4. Dracorhodin Perchlorate Induced Human Breast Cancer MCF-7 Apoptosis through Mitochondrial Pathways

    PubMed Central

    Yu, Jing-hua; Zheng, Gui-bin; Liu, Chun-yu; Zhang, Li-ying; Gao, Hong-mei; Zhang, Ya-hong; Dai, Chun-yan; Huang, Lin; Meng, Xian-ying; Zhang, Wen-yan; Yu, Xiao-fang

    2013-01-01

    Objective: Dracorhodin perchlorate (DP) was a synthetic analogue of the antimicrobial anthocyanin red pigment dracorhodin. It was reported that DP could induce apoptosis in human prostate cancer, human gastric tumor cells and human melanoma, but the cytotoxic effect of DP on human breast cancer was not investigated. This study would investigate whether DP was a candidate chemical of anti-human breast cancer. Methods: The MTT assay reflected the number of viable cells through measuring the activity of cellular enzymes. Phase contrast microscopy visualized cell morphology. Fluorescence microscopy detected nuclear fragmentation after Hoechst 33258 staining. Flowcytometric analysis of Annexin V-PI staining and Rodamine 123 staining was used to detect cell apoptosis and mitochondrial membrane potential (MMP). Real time PCR detected mRNA level. Western blot examined protein expression. Results: DP dose and time-dependently inhibited the growth of MCF-7 cells. DP inhibited MCF-7 cell growth through apoptosis. DP regulated the expression of Bcl-2 and Bax, which were mitochondrial pathway proteins, to decrease MMP, and DP promoted the transcription of Bax and inhibited Bcl-2. Apoptosis-inducing factor (AIF) and cytochrome c which localized in mitochondrial in physiological condition were released into cytoplasm when MMP was decreased. DP activated caspase-9, which was the downstream of mitochondrial pathway. Therefore DP decreased MMP to release AIF and cytochrome c into cytoplasm, further activating caspase 9, lastly led to apoptosis. Conclusion: Therefore DP was a candidate for anti-breast cancer, DP induced apoptosis of MCF-7 through mitochondrial pathway. PMID:23869191

  5. Putting the pieces together: How is the mitochondrial pathway of apoptosis regulated in cancer and chemotherapy?

    PubMed Central

    2014-01-01

    In order to solve a jigsaw puzzle, one must first have the complete picture to logically connect the pieces. However, in cancer biology, we are still gaining an understanding of all the signaling pathways that promote tumorigenesis and how these pathways can be pharmacologically manipulated by conventional and targeted therapies. Despite not having complete knowledge of the mechanisms that cause cancer, the signaling networks responsible for cancer are becoming clearer, and this information is serving as a solid foundation for the development of rationally designed therapies. One goal of chemotherapy is to induce cancer cell death through the mitochondrial pathway of apoptosis. Within this review, we present the pathways that govern the cellular decision to undergo apoptosis as three distinct, yet connected puzzle pieces: (1) How do oncogene and tumor suppressor pathways regulate apoptosis upstream of mitochondria? (2) How does the B-cell lymphoma 2 (BCL-2) family influence tumorigenesis and chemotherapeutic responses? (3) How is post-mitochondrial outer membrane permeabilization (MOMP) regulation of cell death relevant in cancer? When these pieces are united, it is possible to appreciate how cancer signaling directly impacts upon the fundamental cellular mechanisms of apoptosis and potentially reveals novel pharmacological targets within these pathways that may enhance chemotherapeutic success. PMID:25621172

  6. The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

    PubMed Central

    Wenz, Lena-Sophie; Opaliński, Łukasz; Schuler, Max-Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; van der Laan, Martin; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

    2014-01-01

    The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane. PMID:24781695

  7. The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway

    PubMed Central

    Rauthan, Manish; Ranji, Parmida; Aguilera Pradenas, Nataly; Pitot, Christophe; Pilon, Marc

    2013-01-01

    Statins are cholesterol-lowering drugs that inhibit 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the synthesis of cholesterol via the mevalonate pathway. This pathway also produces coenzyme Q (a component of the respiratory chain), dolichols (important for protein glycosylation), and isoprenoids (lipid moieties responsible for the membrane association of small GTPases). We previously showed that the nematode Caenorhabditis elegans is useful to study the noncholesterol effects of statins because its mevalonate pathway lacks the sterol synthesis branch but retains all other branches. Here, from a screen of 150,000 mutagenized genomes, we isolated four C. elegans mutants resistant to statins by virtue of gain-of-function mutations within the first six amino acids of the protein ATFS-1, the key regulator of the mitochondrial unfolded protein response that includes activation of the chaperones HSP-6 and HSP-60. The atfs-1 gain-of-function mutants are also resistant to ibandronate, an inhibitor of an enzyme downstream of HMG-CoA reductase, and to gliotoxin, an inhibitor acting on a subbranch of the pathway important for protein prenylation, and showed improved mitochondrial function and protein prenylation in the presence of statins. Additionally, preinduction of the mitochondrial unfolded protein response in wild-type worms using ethidium bromide or paraquat triggered statin resistance, and similar observations were made in Schizosaccharomyces pombe and in a mammalian cell line. We conclude that statin resistance through maintenance of mitochondrial homeostasis is conserved across species, and that the cell-lethal effects of statins are caused primarily through impaired protein prenylation that results in mitochondria dysfunction. PMID:23530189

  8. N-Methyl, N-propynyl-2-phenylethylamine (MPPE), a Selegiline Analog, Attenuates MPTP-induced Dopaminergic Toxicity with Guaranteed Behavioral Safety: Involvement of Inhibitions of Mitochondrial Oxidative Burdens and p53 Gene-elicited Pro-apoptotic Change.

    PubMed

    Shin, Eun-Joo; Nam, Yunsung; Lee, Ji Won; Nguyen, Phuong-Khue Thi; Yoo, Ji Eun; Tran, The-Vinh; Jeong, Ji Hoon; Jang, Choon-Gon; Oh, Young J; Youdim, Moussa B H; Lee, Phil Ho; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2016-11-01

    Selegiline is a monoamine oxidase-B (MAO-B) inhibitor with anti-Parkinsonian effects, but it is metabolized to amphetamines. Since another MAO-B inhibitor N-Methyl, N-propynyl-2-phenylethylamine (MPPE) is not metabolized to amphetamines, we examined whether MPPE induces behavioral side effects and whether MPPE affects dopaminergic toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Multiple doses of MPPE (2.5 and 5 mg/kg/day) did not show any significant locomotor activity and conditioned place preference, whereas selegiline (2.5 and 5 mg/kg/day) significantly increased these behavioral side effects. Treatment with MPPE resulted in significant attenuations against decreases in mitochondrial complex I activity, mitochondrial Mn-SOD activity, and expression induced by MPTP in the striatum of mice. Consistently, MPPE significantly attenuated MPTP-induced oxidative stress and MPPE-mediated antioxidant activity appeared to be more pronounced in mitochondrial-fraction than in cytosolic-fraction. Because MPTP promoted mitochondrial p53 translocation and p53/Bcl-xL interaction, it was also examined whether mitochondrial p53 inhibitor pifithrin-μ attenuates MPTP neurotoxicity. MPPE, selegiline, or pifithrin-μ significantly attenuated mitochondrial p53/Bcl-xL interaction, impaired mitochondrial transmembrane potential, cytosolic cytochrome c release, and cleaved caspase-3 in wild-type mice. Subsequently, these compounds significantly ameliorated MPTP-induced motor impairments. Neuroprotective effects of MPPE appeared to be more prominent than those of selegiline. MPPE or selegiline did not show any additional protective effects against the attenuation by p53 gene knockout, suggesting that p53 gene is a critical target for these compounds. Our results suggest that MPPE possesses anti-Parkinsonian potentials with guaranteed behavioral safety and that the underlying mechanism of MPPE requires inhibition of mitochondrial oxidative stress, mitochondrial

  9. Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane

    PubMed Central

    Westphal, Dana; Dewson, Grant; Menard, Marie; Frederick, Paul; Iyer, Sweta; Bartolo, Ray; Gibson, Leonie; Czabotar, Peter E.; Smith, Brian J.; Adams, Jerry M.; Kluck, Ruth M.

    2014-01-01

    The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores. PMID:25228770

  10. Reductive stress impairs myoblasts mitochondrial function and triggers mitochondrial hormesis.

    PubMed

    Singh, François; Charles, Anne-Laure; Schlagowski, Anna-Isabel; Bouitbir, Jamal; Bonifacio, Annalisa; Piquard, François; Krähenbühl, Stephan; Geny, Bernard; Zoll, Joffrey

    2015-07-01

    Even though oxidative stress damage from excessive production of ROS is a well known phenomenon, the impact of reductive stress remains poorly understood. This study tested the hypothesis that cellular reductive stress could lead to mitochondrial malfunction, triggering a mitochondrial hormesis (mitohormesis) phenomenon able to protect mitochondria from the deleterious effects of statins. We performed several in vitro experiments on L6 myoblasts and studied the effects of N-acetylcysteine (NAC) at different exposure times. Direct NAC exposure (1mM) led to reductive stress, impairing mitochondrial function by decreasing maximal mitochondrial respiration and increasing H₂O₂production. After 24h of incubation, the reactive oxygen species (ROS) production was increased. The resulting mitochondrial oxidation activated mitochondrial biogenesis pathways at the mRNA level. After one week of exposure, mitochondria were well-adapted as shown by the decrease of cellular ROS, the increase of mitochondrial content, as well as of the antioxidant capacities. Atorvastatin (ATO) exposure (100μM) for 24h increased ROS levels, reduced the percentage of live cells, and increased the total percentage of apoptotic cells. NAC exposure during 3days failed to protect cells from the deleterious effects of statins. On the other hand, NAC pretreatment during one week triggered mitochondrial hormesis and reduced the deleterious effect of statins. These results contribute to a better understanding of the redox-dependant pathways linked to mitochondria, showing that reductive stress could trigger mitochondrial hormesis phenomenon.

  11. Polyunsaturated fatty acids trigger apoptosis of colon cancer cells through a mitochondrial pathway

    PubMed Central

    Zhang, Chengcheng; Yu, Haining; Shen, Yuzhen; Ni, Xiaofeng; Das, Undurti N.

    2015-01-01

    Introduction Colorectal cancer is common in developed countries. Polyunsaturated fatty acids (PUFAs) have been reported to possess tumoricidal action, but the exact mechanism of their action is not clear. Material and methods In the present study, we studied the effect of various n-6 and n-3 fatty acids on the survival of the colon cancer cells LoVo and RKO and evaluated the possible involvement of a mitochondrial pathway in their ability to induce apoptosis. Results It was observed that n-3 α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid (ALA, EPA and DHA respectively) and n-6 linoleic acid, gamma-linolenic acid and arachidonic acid (LA, GLA and AA respectively) induced apoptosis of the colon cancer cells LoVo and RKO at concentrations above 120 μM (p < 0.01 compared to control). The semi-differentiated colon cancer cell line RKO was more sensitive to the cytotoxic action of PUFAs compared to the undifferentiated colon cancer cell line LoVo. PUFA-treated cells showed an increased number of lipid droplets in their cytoplasm. PUFA-induced apoptosis of LoVo and RKO cells is mediated through a mitochondria-mediated pathway as evidenced by loss of mitochondrial membrane potential, generation of ROS, accumulation of intracellular Ca2+, activation of caspase-9 and caspase-3, decreased ATP level and increase in the Bax/Bcl2 expression ratio. Conclusions PUFAs induced apoptosis of colon cancer cells through a mitochondrial dependent pathway. PMID:26528354

  12. Attenuation of Aβ{sub 25–35}-induced parallel autophagic and apoptotic cell death by gypenoside XVII through the estrogen receptor-dependent activation of Nrf2/ARE pathways

    SciTech Connect

    Meng, Xiangbao; Wang, Min; Sun, Guibo; Ye, Jingxue; Zhou, Yanhui; Dong, Xi; Wang, Tingting; Lu, Shan; Sun, Xiaobo

    2014-08-15

    Amyloid-beta (Aβ) has a pivotal function in the pathogenesis of Alzheimer's disease. To investigate Aβ neurotoxicity, we used an in vitro model that involves Aβ{sub 25–35}-induced cell death in the nerve growth factor-induced differentiation of PC12 cells. Aβ{sub 25–35} (20 μM) treatment for 24 h caused apoptotic cell death, as evidenced by significant cell viability reduction, LDH release, phosphatidylserine externalization, mitochondrial membrane potential disruption, cytochrome c release, caspase-3 activation, PARP cleavage, and DNA fragmentation in PC12 cells. Aβ{sub 25–35} treatment led to autophagic cell death, as evidenced by augmented GFP-LC3 puncta, conversion of LC3-I to LC3-II, and increased LC3-II/LC3-I ratio. Aβ{sub 25–35} treatment induced oxidative stress, as evidenced by intracellular ROS accumulation and increased production of mitochondrial superoxide, malondialdehyde, protein carbonyl, and 8-OHdG. Phytoestrogens have been proved to be protective against Aβ-induced neurotoxicity and regarded as relatively safe targets for AD drug development. Gypenoside XVII (GP-17) is a novel phytoestrogen isolated from Gynostemma pentaphyllum or Panax notoginseng. Pretreatment with GP-17 (10 μM) for 12 h increased estrogen response element reporter activity, activated PI3K/Akt pathways, inhibited GSK-3β, induced Nrf2 nuclear translocation, augmented antioxidant responsive element enhancer activity, upregulated heme oxygenase 1 (HO-1) expression and activity, and provided protective effects against Aβ{sub 25–35}-induced neurotoxicity, including oxidative stress, apoptosis, and autophagic cell death. In conclusion, GP-17 conferred protection against Aβ{sub 25–35}-induced neurotoxicity through estrogen receptor-dependent activation of PI3K/Akt pathways, inactivation of GSK-3β and activation of Nrf2/ARE/HO-1 pathways. This finding might provide novel insights into understanding the mechanism for neuroprotective effects of phytoestrogens or

  13. A role for transforming growth factor-{beta} apoptotic signaling pathway in liver injury induced by ingestion of water contaminated with high levels of Cr(VI)

    SciTech Connect

    Rafael, A.I.; Almeida, A.; Santos, P.; Parreira, I.; Madeira, V.M.S.; Alves, R.; Cabrita, A.M.S.; Alpoim, M.C.

    2007-10-15

    Hexavalent chromium [Cr(VI)] exposure is commonly associated with lung cancer. Although other adverse health effects have been reported, some authors, on assuming that orally ingested Cr(VI) is efficiently detoxified upon reduction by body fluids, believe that Cr(VI) do not target cells other than respiratory tract cells. In rodents, ingested Cr(VI)-contaminated water was reported to induce, in the liver, increases in TGF-{beta} transcripts. As TGF-{beta} dependent signaling pathways are closely associated with hepatic injury, the present study was undertaken addressing two specific issues: the effects of ingestion of water contaminated with high levels of Cr(VI) in rat liver structure and function; and the role of the TGF-{beta} pathway in Cr(VI)-induced liver injury. Examination of Wistar rats exposed to 20 ppm Cr(VI)-contaminated water for 10 weeks showed increased serum glucose and alanine aminotransferase (ALT) levels. Liver histological examination revealed hepatocellular apoptosis, further confirmed by immunohystochemical study of Caspase 3 expression. Liver gene expression analysis revealed increased expression of Smad2/Smad4 and Dapk, suggesting the involvement of the TGF-{beta} pathway in the apoptotic process. Since no changes in Smad3 expression were observed it appears apoptosis is using a Smad3-independent pathway. Increased expression of both Caspase 8 and Daxx genes suggests also the involvement of the Fas pathway. Gene expression analysis also revealed that a p160{sup ROCK}-Rho-independent pathway operates, leading to cell contraction and membrane blebbing, characteristic apoptotic features. These findings suggest that either the amount of Cr(VI) ingested overwhelmed the body fluids reductive capacity or some Cr(VI) escapes the reductive protection barrier, thus targeting the liver and inducing apoptosis.

  14. Differential induction of apoptosis in human colonic carcinoma cells (Caco-2) by Atopobium, and commensal, probiotic and enteropathogenic bacteria: mediation by the mitochondrial pathway.

    PubMed

    Altonsy, Mohammed O; Andrews, Simon C; Tuohy, Kieran M

    2010-02-28

    The induction of apoptosis in mammalian cells by bacteria is well reported. This process may assist infection by pathogens whereas for non-pathogens apoptosis induction within carcinoma cells protects against colon cancer. Here, apoptosis induction by a major new gut bacterium, Atopobium minutum, was compared with induction by commensal (Escherichia coli K-12 strains), probiotic (Lactobacillus rhamnosus, Bifidobacterium latis) and pathogenic (E. coli: EPEC and VTEC) gut bacteria within the colon cancer cell line, Caco-2. The results show a major apoptotic effect for the pathogens, mild effects for the probiotic strains and A. minutum, but no effect for commensal E. coli. The mild apoptotic effects observed are consistent with the beneficial roles of probotics in protection against colon cancer and suggest, for the first time, that A. minutum possesses similar advantageous, anti-cancerous activity. Although bacterial infection increased Caco-2 membrane FAS levels, caspase-8 was not activated indicating that apoptosis is FAS independent. Instead, in all cases, apoptosis was induced through the mitochondrial pathway as indicated by BAX translocation, cytochrome c release, and caspase-9 and -3 cleavage. This suggests that an intracellular stimulus initiates the observed apoptosis responses.

  15. Acidic polysaccharide of Panax ginseng regulates the mitochondria/caspase-dependent apoptotic pathway in radiation-induced damage to the jejunum in mice.

    PubMed

    Bing, So Jin; Kim, Min Ju; Ahn, Ginnae; Im, Jaehak; Kim, Dae Seung; Ha, Danbee; Cho, Jinhee; Kim, Areum; Jee, Youngheun

    2014-04-01

    Owing to its susceptibility to radiation, the small intestine of mice is valuable for studying radioprotective effects. When exposed to radiation, intestinal crypt cells immediately go through apoptosis, which impairs swift differentiation necessary for the regeneration of intestinal villi. Our previous studies have elucidated that acidic polysaccharide of Panax ginseng (APG) protects the mouse small intestine from radiation-induced damage by lengthening villi with proliferation and repopulation of crypt cells. In the present study, we identified the molecular mechanism involved. C57BL/6 mice were irradiated with gamma-rays with or without APG and the expression levels of apoptosis-related molecules in the jejunum were investigated using immunohistochemistry. APG pretreatment strongly decreased the radiation-induced apoptosis in the jejunum. It increased the expression levels of anti-apoptotic proteins (Bcl-2 and Bcl-XS/L) and dramatically reduced the expression levels of pro-apoptotic proteins (p53, BAX, cytochrome c and caspase-3). Therefore, APG attenuated the apoptosis through the intrinsic pathway, which is controlled by p53 and Bcl-2 family members. Results presented in this study suggest that APG protects the mouse small intestine from irradiation-induced apoptosis through inhibition of the p53-dependent pathway and the mitochondria/caspase pathway. Thus, APG may be a potential agent for preventing radiation induced injuries in intestinal cells during radio-therapy such as in cancer treatment.

  16. The mitochondrial alternative oxidase pathway protects the photosynthetic apparatus against photodamage in Rumex K-1 leaves

    PubMed Central

    2012-01-01

    Background It is known that excess reducing equivalents in the form of NADPH in chloroplasts can be transported via shuttle machineries, such as the malate-oxaloacetate (OAA) shuttle, into the mitochondria, where they are efficiently oxidised by the mitochondrial alternative oxidase (AOX) respiratory pathway. Therefore, it has been speculated that the AOX pathway may protect plants from photoinhibition, but the mechanism by which this protection occurs remains to be elucidated. Results The observation that the malate-OAA shuttle activity and the AOX pathway capacity increased markedly after intense light treatment in Rumex K-1 leaves indicates that excess NADPH was transported from the chloroplasts and oxidised by the AOX pathway. The inhibition of the AOX pathway by salicylhydroxamic acid (SHAM) caused the over-reduction of the photosystem I (PSI) acceptor side, as indicated by the increases in the extent of reduction of P700+. Furthermore, the photosynthetic linear electron flow was restricted, which was indicated by the decreases in the PSII electron transport rate (ETR) and the photosynthetic O2 evolution rate. The restriction of the photosynthetic linear electron flow, which generates the thylakoid ΔpH, inevitably decreased the de-epoxidation of the xanthophyll cycle (ΔPRI). Therefore, the induction of non-photochemical quenching (NPQ) was suppressed when the AOX pathway was inhibited. The effect of the inhibition of the AOX pathway on NPQ induction was less at 20 mM NaHCO3 than at 1 mM NaHCO3. The suppression of NPQ induction by the inhibition of the AOX pathway was also observed during the induction phase of photosynthesis. In addition, the inhibition of the AOX pathway increased the accumulation of hydrogen peroxide (H2O2), suggesting that the AOX pathway functions as an antioxidant mechanism. Conclusions The inhibition of the AOX pathway resulted in the rapid accumulation of NADPH in the chloroplasts, which caused the over-reduction of the PSI acceptor

  17. Induction of apoptosis in hepatocellular carcinoma Smmc-7721 cells by vitamin K(2) is associated with p53 and independent of the intrinsic apoptotic pathway.

    PubMed

    Li, Lu; Qi, Zhiling; Qian, Jin; Bi, Fuyong; Lv, Jun; Xu, Lei; Zhang, Ling; Chen, Hongyu; Jia, Renbing

    2010-09-01

    Vitamin K(2) (VK(2)) can exert cell growth inhibitory effects in various human cancer cells. In this study, we investigated the cell growth inhibitory effects of VK(2) in hepatocellular carcinoma Smmc-7721 cells and the mechanisms involved. We found that VK(2)-inhibited cell proliferation in Smmc-7721 cells in a dose-dependent manner, and the IC50 of VK(2) in Smmc-7721 cells was 9.73 microM at 24 h. The data from flow cytometric analyses, DNA fragmentation assays, and caspase 3 activity assays revealed that apoptosis was the determining factor in VK(2) activity. Furthermore, a significant increase in p53 phosphorylation and protein level was exhibited in apoptotic cells treated with VK(2), although there were no changes in p53 mRNA expression. Bax expression was unaffected by VK(2) in Smmc-7721 cells. In addition, our study showed that caspase 3 was activated by caspase 8, not caspase 9, in Smmc-7721 cells treated with VK(2). In summary, these data suggested that VK(2) can inhibit the growth of Smmc-7721 cells by induction of apoptosis involving caspase 8 activation and p53. This apoptotic process was not mediated by the intrinsic apoptotic pathway.

  18. Myocardial oxidative damage is induced by cardiac Fas-dependent and mitochondria-dependent apoptotic pathways in human cocaine-related overdose

    PubMed Central

    Turillazzi, Emanuela; Cerretani, Daniela; Cantatore, Santina; Fiaschi, Anna Ida; Frati, Paola; Micheli, Lucia; Neri, Margherita; Cipolloni, Luigi; Di Paolo, Marco; Pinchi, Enrica; Riezzo, Irene; Santurro, Alessandro; Vullo, Annamaria; Fineschi, Vittorio

    2017-01-01

    The aim of this study is to analyse cardiac specimens from human cocaine-related overdose, to verify the hypothesis that cardiac toxicity by acute exposure to high dosage of cocaine could be mediated by unbalanced myocardial oxidative stress, and to evaluate the apoptotic response. To address these issues, biochemical and immunohistological markers of oxidative/nitrosative stress were evaluated. We found that i-NOS, NOX2 and nitrotyrosine expression were significantly higher in the hearts of subjects who had died from high doses of cocaine, compared to the control group. Increase of these markers was associated with a dramatic increase in 8-OHdG, another marker of oxidative stress. A high number of TUNEL-positive apoptotic myocells was observed in the study group compared to the control group. The immunoexpression of TNF-α was significantly higher in the cocaine group compared to the control group. Furthermore, we detected a significantly stronger immunoresponse to anti-SMAC/DIABLO in our study group compared to control cases. Both cardiac Fas-dependent and mitochondria-dependent apoptotic pathways appeared to be activated to a greater extent in the cocaine group than in the control group. Our results highlight the central role of oxidative stress in cocaine toxicity. High levels of NOS can promote the oxidation process and lead to apoptosis. PMID:28281685

  19. Neuroprotective effects of lycopene in spinal cord injury in rats via antioxidative and anti-apoptotic pathway.

    PubMed

    Hu, Wei; Wang, Hongbo; Liu, Zhenfeng; Liu, Yanlu; Wang, Rong; Luo, Xiao; Huang, Yifei

    2017-03-06

    Oxidative damage induced-mitochondrial dysfunction and apoptosis has been widely studied in spinal cord injury (SCI). Lycopene, a polyunsaturated hydrocarbon, has the highest antioxidant capacity compared to the other carotenoids. However, the role of lycopene in SCI is unknown. In the present study, we evaluated the antioxidant effects of lycopene on mitochondrial dysfunction and apoptosis following T10 contusion SCI in rats. The rats were randomized into 5 groups: the sham group, the SCI group and the SCI pre-treated with lycopene (5, 10, or 20mg/kg) group. The SCI group showed increased malondialdehyde (MDA) content, decreased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) ability, which indicated that SCI could induce oxidative damage. What's more, the SCI group showed decreased mRNA expression of cytochrome b and mitochondrial transcription factor A (Tfam), and decreased mitochondrial membrane potential (ΔYm), which indicated that SCI could induce mitochondrial dysfunction. Besides, the SCI group showed decreased protein expression of bcl-2 and mitochondrial cytochrome C, increased protein expression of cytosolic cytochrome C, cleaved caspase-9, cleaved caspase-3 and bax, and increased TUNEL-positive cell numbers, which indicated that SCI could induce cell apoptosis. Fortunately, the lycopene treatment significantly ameliorated oxidative damage, mitochondrial dysfunction and cell apoptosis via the reversion of those parameters described above in the dose of lycopene of 10 and 20mg/kg. In addition, lycopene significantly ameliorated the hind limb motor disturbances in the SCI+lyco10 group and the SCI+lyco20 group compared with the SCI group. These results suggested that lycopene administration could improve total antioxidant status and might have neuroprotective effects on SCI.

  20. Noscapine Increases the Sensitivity of Drug-Resistant Ovarian Cancer Cell Line SKOV3/DDP to Cisplatin by Regulating Cell Cycle and Activating Apoptotic Pathways.

    PubMed

    Shen, Wei; Liang, Bingfeng; Yin, Jie; Li, Xiurong; Cheng, Jianxin

    2015-05-01

    Cisplatin is a first-line chemotherapy drug against ovarian cancer. However, its strong toxic side effects and the development of cisplatin resistance in human cancer cells seriously influence the effects of chemotherapy and quality of life in patients. Noscapine (Nos), a non-toxic benzylisoquinoline alkaloid extracted from opium, has been recently reported to have anti-cancer activity, but the mechanism of that effect has not been clearly established. In the present study, we investigated cytotoxicity of Nos in combination with cisplatin (DDP) in drug-resistant human ovarian cancer cell line SKOV3/DDP in vitro and in vivo null mice xenograft model. Cell proliferation was measured by MTT assay, flow cytometry was used to analyze cell cycle and apoptosis, protein expression of several apoptotic factors was investigated by flow cytometry and immunohistochemical method, and their mRNA expression levels were determined by real-time PCR. In vitro experiments showed that Nos significantly inhibited proliferation of SKOV3/DDP cells. DDP/Nos-combined treatment notably enhanced DDP-induced inhibition of cell proliferation and increased the pro-apoptotic effect of DDP in SKOV3/DDP cells. DDP/Nos administration increased the proportion of G2/M cells, reduced both protein and mRNA expression of anti-apoptotic factors XIAP, surviving and NF-kB, and augmented protein and mRNA levels of pro-apoptotic caspase-3. In vivo experiments revealed that Nos/DDP treatment increased the apoptotic rate of xenograft tumors in null mice. Tumor volume decreased from 1.733 ± 0.155 g in mice treated with DDP alone to 1.191 ± 0.106 g in animals treated with Nos/DDP. These observations suggest that Nos increases the anti-cancer activity of DDP against the drug-resistant ovarian cancer cell line SKOV3/DDP by modulating the cell cycle and activating apoptotic pathways. The study provides a new chemotherapy strategy for the treatment of DDP-resistant human ovarian cancer.

  1. Slm35 links mitochondrial stress response and longevity through TOR signaling pathway

    PubMed Central

    Jose, L. Aguilar-Lopez; Laboy, Raymond; Fabiola, Jaimes-Miranda; Garay, Erika; Alexander, DeLuna; Funes, Soledad

    2016-01-01

    In most eukaryotic cells mitochondria are essential organelles involved in a great variety of cellular functions. One of the physiological processes linked to mitochondria is aging, a gradual process of damage accumulation that eventually promotes cell death. Aging depends on a balance between mitochondrial biogenesis, function and degradation. It has been previously shown that Tor1, Sch9 and Ras2 are activated in response to nutrient availability and regulate cell growth and division. A deficiency in any of these genes promotes lifespan extension and cell protection during oxidative and heat shock stress. In this work we report that in Saccharomyces cerevisiae, the uncharacterized mitochondrial protein Slm35 is functionally linked with the TOR signaling pathway. A Δtor1Δslm35 strain shows a severe decrease in lifespan and is unable to contend with oxidative and heat shock stresses. Specifically, this mutant shows decreased catalase activity indicating a misregulation of ROS scavenging mechanisms. In this study we show that Slm35 is also relevant for mitochondrial network dynamics and mitophagy. The results presented here suggest that Slm35 plays an important role connecting mitochondrial function with cytosolic responses and cell adaptation to stress and aging. PMID:27922823

  2. Slm35 links mitochondrial stress response and longevity through TOR signaling pathway.

    PubMed

    Aguilar-Lopez, Jose L; Laboy, Raymond; Jaimes-Miranda, Fabiola; Garay, Erika; DeLuna, Alexander; Funes, Soledad

    2016-12-02

    In most eukaryotic cells mitochondria are essential organelles involved in a great variety of cellular functions. One of the physiological processes linked to mitochondria is aging, a gradual process of damage accumulation that eventually promotes cell death. Aging depends on a balance between mitochondrial biogenesis, function and degradation. It has been previously shown that Tor1, Sch9 and Ras2 are activated in response to nutrient availability and regulate cell growth and division. A deficiency in any of these genes promotes lifespan extension and cell protection during oxidative and heat shock stress. In this work we report that in Saccharomyces cerevisiae, the uncharacterized mitochondrial protein Slm35 is functionally linked with the TOR signaling pathway. A Δtor1Δslm35 strain shows a severe decrease in lifespan and is unable to contend with oxidative and heat shock stresses. Specifically, this mutant shows decreased catalase activity indicating a misregulation of ROS scavenging mechanisms. In this study we show that Slm35 is also relevant for mitochondrial network dynamics and mitophagy. The results presented here suggest that Slm35 plays an important role connecting mitochondrial function with cytosolic responses and cell adaptation to stress and aging.

  3. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death.

    PubMed

    Zaja, Ivan; Bai, Xiaowen; Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G; Bosnjak, Zeljko J

    2014-10-31

    Myocardial ischemia-reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of mitochondrial fission; and (2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.

  4. An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation

    PubMed Central

    Harner, Max E; Unger, Ann-Katrin; Geerts, Willie JC; Mari, Muriel; Izawa, Toshiaki; Stenger, Maria; Geimer, Stefan; Reggiori, Fulvio; Westermann, Benedikt; Neupert, Walter

    2016-01-01

    Metabolic function and architecture of mitochondria are intimately linked. More than 60 years ago, cristae were discovered as characteristic elements of mitochondria that harbor the protein complexes of oxidative phosphorylation, but how cristae are formed, remained an open question. Here we present experimental results obtained with yeast that support a novel hypothesis on the existence of two molecular pathways that lead to the generation of lamellar and tubular cristae. Formation of lamellar cristae depends on the mitochondrial fusion machinery through a pathway that is required also for homeostasis of mitochondria and mitochondrial DNA. Tubular cristae are formed via invaginations of the inner boundary membrane by a pathway independent of the fusion machinery. Dimerization of the F1FO-ATP synthase and the presence of the MICOS complex are necessary for both pathways. The proposed hypothesis is suggested to apply also to higher eukaryotes, since the key components are conserved in structure and function throughout evolution. DOI: http://dx.doi.org/10.7554/eLife.18853.001 PMID:27849155

  5. Evidence of an alternative oxidase pathway for mitochondrial respiration in the scuticociliate Philasterides dicentrarchi.

    PubMed

    Mallo, Natalia; Lamas, Jesús; Leiro, José Manuel

    2013-11-01

    The presence of an alternative oxidase (AOX) in the mitochondria of the scuticociliate P. dicentrarchi was investigated. The mitochondrial oxygen consumption was measured in the presence of KCN, an inhibitor of cytochrome pathway (CP) respiration and salicylhydroxamic acid (SHAM), a specific inhibitor of alternative pathway (AP) respiration. AOX expression was monitored by western blotting with an AOX polyclonal antibody. The results showed that P. dicentrarchi possesses a branched mitochondrial electron transport chain with both cyanide-sensitive and -insensitive oxygen consumption. Mitochondrial respiration was partially inhibited by cyanide and completely inhibited by the combination of cyanide and SHAM, which is direct evidence for the existence of an AP in this ciliate. SHAM significantly inhibited in vitro growth of trophozoites both under normoxic and hypoxic conditions. AOX is a 42kD monomeric protein inducible by hypoxic conditions in experimental infections and by CP inhibitors such as cyanide and antimycin A, or by AP inhibitors such as SHAM. CP respiration was greatly stimulated during the exponential growth phase, while AP respiration increased during the stationary phase, in which AOX expression is induced. As the host does not possess AOX, and because during infection P. dicentrarchi respires via AP, it may be possible to develop inhibitors targeting the AP as a novel anti-scuticociliate therapy.

  6. Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways

    PubMed Central

    Xiong, Xin-xin; Liu, Ju-mei; Qiu, Xin-yao; Pan, Feng; Yu, Shang-bin; Chen, Xiao-qian

    2015-01-01

    Aim: To investigate the effects of piperlongumine (PL), an anticancer alkaloid from long pepper plants, on the primary myeloid leukemia cells from patients and the mechanisms of action. Methods: Human BM samples were obtained from 9 patients with acute or chronic myeloid leukemias and 2 patients with myelodysplastic syndrome (MDS). Bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Cell viability was determined using MTT assay, and apoptosis was examined with PI staining or flow cytometry. ROS levels in the cells were determined using DCFH-DA staining and flow cytometry. Expression of apoptotic and autophagic signaling proteins was analyzed using Western blotting. Results: PL inhibited the viability of BMMNCs from the patients with myeloid leukemias (with IC50 less than 20 μmol/L), but not that of BMMNCs from a patient with MDS. Furthermore, PL (10 and 20 μmol/L) induced apoptosis of BMMNCs from the patients with myeloid leukemias in a dose-dependent manner. PL markedly increased ROS levels in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the antioxidant N-acetyl-L-cysteine abolished PL-induced ROS accumulation and effectively reduced PL-induced cytotoxicity. Moreover, PL markedly increased the expression of the apoptotic proteins (Bax, Bcl-2 and caspase-3) and autophagic proteins (Beclin-1 and LC3B), and phosphorylation of p38 and JNK in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the specific p38 inhibitor SB203580 or the specific JNK inhibitor SP600125 partially reversed PL-induced ROS production, apoptotic/autophagic signaling activation and cytotoxicity. Conclusion: Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways. PMID:25619389

  7. Ethanolic extract of fermented Thunb induces human leukemic HL-60 and Molt-4 cell apoptosis via oxidative stress and a mitochondrial pathway.

    PubMed

    Banjerdpongchai, Ratana; Kongtawelert, Prachya

    2011-01-01

    Houttuynia cordata Thunb (HCT) is a medicinal plant of the Saururaceae family which features antimutagenic and antiviral properties. For extraction, the whole plants were fermented or non-fermented with yeast and ethanol then the whole plants were dried, ground and extracted with 95% ethanol or water. The aims of this study were to compare cytotoxic effects, apoptosis induction, and mechanism(s) with the ethanolic and water extracts of fermented and non-fermented HCT. Cytotoxicity was assessed using the MTT assay in human leukemic HL-60, Molt-4 and peripheral blood mononuclear cells (PBMCs). Apoptotic death was characterized by staining with propidium iodide and examined under a fluorescence microscope. Peroxide radical production and reduction of mitochondrial transmembrane potential (MTP) were determined using 2',7'-dichlorohydrofluorescein diacetate and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. The expression of caspase-9 was identified by immunoblotting. The ethanolic extract of fermented HCT was cytotoxic to HL-60 >Molt- 4 > PBMCs, to a greater extent than the non-fermented preparation and the number of apoptotic cells was higher. The alcoholic (fermented) extract produced more radicals than the non-fermented in HL-60 cells but the converse was observed in Molt-4 cells. Reduction of MTP was found in HL-60 and Molt-4 cells treated with the alcoholic (fermented) extract and caspase-9 was cleaved dose-dependently in both cells. In conclusion, the alcoholic extract of fermented HCT was more toxic to human leukemic cells than the non-fermented and both cell lines underwent apoptosis via oxidative stress and a mitochondrial pathway.

  8. Fenugreek, a naturally occurring edible spice, kills MCF-7 human breast cancer cells via an apoptotic pathway.

    PubMed

    Khoja, Kholoud K; Shaf, Gowhar; Hasan, Tarique N; Syed, Naveed Ahmed; Al-Khalifa, Abdrohman S; Al-Assaf, Abdullah H; Alshatwi, Ali A

    2011-01-01

    There is growing use of anticancer complementary and alternative medicines worldwide. Trigonella foenum graecum (Fenugreek) is traditionally applied to treat disorders such as diabetes, high cholesterol, wounds, inflammation, and gastrointestinal ailments. Fenugreek is also reported to have anticancer properties due to its active beneficial chemical constituents. The mechanism of action of several anticancer drugs is based on their ability to induce apoptosis. The objective of the study was to characterize the downstream apoptotic genes targeted by FCE in MCF-7 human immortalized breast cells. FCE effectively killed MCF-7 cells through induction of apoptosis,confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and RT-PCR assays. When cells were exposed to 50 μg/mL FCE for 24 hours, 23.2% apoptotic cells resulted, while a 48-hour exposure to 50 μg/mL caused 73.8% apoptosis. This was associated with increased expression of Caspase 3, 8, 9, p53, Fas, FADD, Bax and Bak in a time-and dose-dependent manner, as determined by real- time quantitative PCR. In summary, the induction of apoptosis by FCE is effected by its ability to increase the expression of pro-apoptotic genes and the spice holds promise for consideration in complementary therapy for breast cancer patients.

  9. Role of type-II pathway in apoptotic cell death induction by photosensitized CDRI-97/78 under ambient exposure of UV-B.

    PubMed

    Dwivedi, Ashish; Pal, Manish Kumar; Tripathi, Amit Kumar; Yadav, Neera; Mujtaba, Syed Faiz; Pant, M C; Singh, Shio Kumar; Mishra, Durga Prasad; Ray, Ratan Singh; Manjunatha Prabhu, B H

    2013-10-24

    Novel trioxane 97/78, developed by Central Drug Research Institute (CDRI), Lucknow has shown promising antimalarial activity. Clinical experience of anti-malarial drugs registered the occurrence of phototoxicity in patients exposed with sunlight subsequent to medication. Photodegradation study has identified one photo-product up to 4h under UV-B/Sunlight by LC-MS/MS. UV-B irradiated 97/78 compound produced ¹O₂ via type-II dependent reaction mechanism, corroborated by its specific quencher. 2'-dGuO degradation and % tail development in photochemical as well as comet test, advocated the genotoxic potential of 97/78. The photocytotoxicity assays (MTT and NRU) on HaCaT cell line revealed the considerable decline in cell viability by 97/78. Cell cycle and Annexin V/PI double stain along with AO/EB demonstrated the G2/M phase arrest and apoptosis. Significant caspase-3 activity was measured in photoexcited 97/78 by colorimetric assay. Fluorescence stain with AO/JC-1 confirmed the lysosomal disruption and mitochondrial membrane destabilization by UV-B irradiated 97/78. Gene expression by RT-PCR showed significant upregulation of p21 and pro-apoptotic Bax, but no change observed in Bcl-2. In conclusion, the study highlights ROS mediated DNA damage, lysosomal and mitochondrial destabilization via upregulation of Bax and activation of caspase-3 which further leads to apoptosis.

  10. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

    SciTech Connect

    Zaja, Ivan; Bai, Xiaowen; Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G.; Bosnjak, Zeljko J.

    2014-10-31

    Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

  11. Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway

    NASA Astrophysics Data System (ADS)

    Zeng, Chuan-Chuan; Zhang, Cheng; Yao, Jun-Hua; Lai, Shang-Hai; Han, Bing-Jie; Li, Wei; Tang, Bing; Wan, Dan; Liu, Yun-Jun

    2016-11-01

    In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5 ± 1.2 μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

  12. Econazole Nitrate Induces Apoptosis in MCF-7 Cells via Mitochondrial and Caspase Pathways

    PubMed Central

    Sun, Juan; Yu, Chun-Hui; Zhao, Xue-Ling; Wang, Yang; Jiang, Shou-Gang; Gong, Xian-Feng

    2014-01-01

    Econazole nitrate (EN), a synthetic compound, is now in use as a routine antifungal drug. EN was shown to have antitumor effect, the tumor cell killing mechanisms, however, remain unclear. In this research, the apoptosis-inducing effect of EN on MCF-7 cells was investigated. The results showed that EN inhibited the proliferation of MCF-7 cells in a time- and dose-dependent manner by MTT method and colony forming assay. MCF-7 cells treated with EN showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Meanwhile, the loss of mitochondrial membrane potential was showed by flow cytometry. In addition, western blot analysis showed that EN resulted in the decrease expression of procaspase-3, procaspase-9 and bcl-2. In conclusion, these findings suggest that EN may be an effective way for treating human breast cancer. The anti-tumor mechanisms of EN might involve mitochondrial and caspase pathways. PMID:25587322

  13. The complexity of apoptotic cell death in mollusks: An update.

    PubMed

    Romero, A; Novoa, B; Figueras, A

    2015-09-01

    Apoptosis is a type of programmed cell death that produces changes in cell morphology and in biochemical intracellular processes without inflammatory reactions. The components of the apoptotic pathways are conserved throughout evolution. Caspases are key molecules involved in the transduction of the death signal and are responsible for many of the biochemical and morphological changes associated with apoptosis. Nowadays, It is known that caspases are activated through two major apoptotic pathways (the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway), but there are also evidences of at least other alternative pathway (the perforin/granzyme pathway). Apoptosis in mollusks seems to be similar in complexity to apoptosis in vertebrates but also has unique features maybe related to their recurrent exposure to environmental changes, pollutants, pathogens and also related to the sedentary nature of some stages in the life cycle of mollusks bivalves and gastropods. As in other animals, apoptotic process is involved in the maintenance of tissue homeostasis and also constitutes an important immune response that can be triggered by a variety of stimuli, including cytokines, hormones, toxic insults, viruses, and protozoan parasites. The main goal of this work is to present the current knowledge of the molecular mechanisms of apoptosis in mollusks and to highlight those steps that need further study.

  14. Targeting RING domains of Mdm2-MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells.

    PubMed

    Wu, W; Xu, C; Ling, X; Fan, C; Buckley, B P; Chernov, M V; Ellis, L; Li, F; Muñoz, I G; Wang, X

    2015-12-31

    Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

  15. Cordyceps militaris induces tumor cell death via the caspase‑dependent mitochondrial pathway in HepG2 and MCF‑7 cells.

    PubMed

    Song, Jingjing; Wang, Yingwu; Teng, Meiyu; Zhang, Shiqiang; Yin, Mengya; Lu, Jiahui; Liu, Yan; Lee, Robert J; Wang, Di; Teng, Lesheng

    2016-06-01

    Cordyceps militaris (CM), an entomopathogenic fungus belonging to the class ascomycetes, possesses various pharmacological activities, including cytotoxic effects, on various types of human tumor cells. The present study investigated the anti‑hepatocellular carcinoma (HCC) and anti‑breast cancer effects of CM in in vitro and in vivo models. CM aqueous extract reduced cell viability, suppressed cell proliferation, inhibited cell migration ability, caused the over-release of lactate dehydrogenase, induced mitochondrial dysfunction and enhanced apoptotic rates in MCF‑7 and HepG2 cells. The expression levels of cleaved poly (ADP ribose) polymerase and caspase‑3, biomarkers of apoptosis, were increased following treatment with CM aqueous extract for 24 h. Furthermore, in the MCF‑7 and HepG2 cells, enhanced levels of B cell‑associated X protein and cleaved caspase‑8 were observed in the CM‑treated cells. Finally, the antitumor activities of CM in HCC and breast cancer were also confirmed in MCF‑7‑ and HepG2‑xengraft nude mice models. Collectively, the data obtained in the present study suggested that the cytotoxic effects of CM aqueous extract on HCC and breast cancer are associated with the caspase‑dependent mitochondrial pathway.

  16. Cordyceps militaris induces tumor cell death via the caspase-dependent mitochondrial pathway in HepG2 and MCF-7 cells

    PubMed Central

    SONG, JINGJING; WANG, YINGWU; TENG, MEIYU; ZHANG, SHIQIANG; YIN, MENGYA; LU, JIAHUI; LIU, YAN; LEE, ROBERT J; WANG, DI; TENG, LESHENG

    2016-01-01

    Cordyceps militaris (CM), an entomopathogenic fungus belonging to the class ascomycetes, possesses various pharmacological activities, including cytotoxic effects, on various types of human tumor cells. The present study investigated the anti-hepatocellular carcinoma (HCC) and anti-breast cancer effects of CM in in vitro and in vivo models. CM aqueous extract reduced cell viability, suppressed cell proliferation, inhibited cell migration ability, caused the over-release of lactate dehydrogenase, induced mitochondrial dysfunction and enhanced apoptotic rates in MCF-7 and HepG2 cells. The expression levels of cleaved poly (ADP ribose) polymerase and caspase-3, biomarkers of apoptosis, were increased following treatment with CM aqueous extract for 24 h. Furthermore, in the MCF-7 and HepG2 cells, enhanced levels of B cell-associated X protein and cleaved caspase-8 were observed in the CM-treated cells. Finally, the antitumor activities of CM in HCC and breast cancer were also confirmed in MCF-7- and HepG2-xengraft nude mice models. Collectively, the data obtained in the present study suggested that the cytotoxic effects of CM aqueous extract on HCC and breast cancer are associated with the caspase-dependent mitochondrial pathway. PMID:27109250

  17. Regulation of signal transducer and activator of transcription 3 and apoptotic pathways by betaine attenuates isoproterenol-induced acute myocardial injury in rats.

    PubMed

    Zheng, P; Liu, J; Mai, S; Yuan, Y; Wang, Y; Dai, G

    2015-05-01

    The present study was designed to investigate the cardioprotective effects of betaine on acute myocardial ischemia induced experimentally in rats focusing on regulation of signal transducer and activator of transcription 3 (STAT3) and apoptotic pathways as the potential mechanism underlying the drug effect. Male Sprague Dawley rats were treated with betaine (100, 200, and 400 mg/kg) orally for 40 days. Acute myocardial ischemic injury was induced in rats by subcutaneous injection of isoproterenol (85 mg/kg), for two consecutive days. Serum cardiac marker enzyme, histopathological variables and expression of protein levels were analyzed. Oral administration of betaine (200 and 400 mg/kg) significantly reduced the level of cardiac marker enzyme in the serum and prevented left ventricular remodeling. Western blot analysis showed that isoproterenol-induced phosphorylation of STAT3 was maintained or further enhanced by betaine treatment in myocardium. Furthermore, betaine (200 and 400 mg/kg) treatment increased the ventricular expression of Bcl-2 and reduced the level of Bax, therefore causing a significant increase in the ratio of Bcl-2/Bax. The protective role of betaine on myocardial damage was further confirmed by histopathological examination. In summary, our results showed that betaine pretreatment attenuated isoproterenol-induced acute myocardial ischemia via the regulation of STAT3 and apoptotic pathways.

  18. Lycopene protects human SH-SY5Y neuroblastoma cells against hydrogen peroxide-induced death via inhibition of oxidative stress and mitochondria-associated apoptotic pathways

    PubMed Central

    FENG, CHUNSHENG; LUO, TIANFEI; ZHANG, SHUYAN; LIU, KAI; ZHANG, YANHONG; LUO, YINAN; GE, PENGFEI

    2016-01-01

    Oxidative stress, which is characterized by excessive production of reactive oxygen species (ROS), is a common pathway that results in neuronal injury or death due to various types of pathological stress. Although lycopene has been identified as a potent antioxidant, its effect on hydrogen peroxide (H2O2)-induced neuronal damage remains unclear. In the present study, pretreatment with lycopene was observed to protect SH-SY5Y neuroblastoma cells against H2O2-induced death via inhibition of apoptosis resulting from activation of caspase-3 and translocation of apoptosis inducing factor (AIF) to the nucleus. Furthermore, the over-produced ROS, as well as the reduced activities of anti-oxidative enzymes, superoxide dismutase and catalase, were demonstrated to be alleviated by lycopene. Additionally, lycopene counteracted H2O2-induced mitochondrial dysfunction, which was evidenced by suppression of mitochondrial permeability transition pore opening, attenuation of the decline of the mitochondrial membrane potential, and inhibition of the increase of Bax and decrease of Bcl-2 levels within the mitochondria. The release of cytochrome c and AIF from the mitochondria was also reduced. These results indicate that lycopene is a potent neuroprotectant against apoptosis, oxidative stress and mitochondrial dysfunction, and could be administered to prevent neuronal injury or death. PMID:27035331

  19. [Role of mitochondrial alternative oxidase (AOX) pathway in photoprotection in Rumex K-1 leaves].

    PubMed

    Meng, Xiang-Long; Zhang, Li-Tao; Zhang, Zi-Shan; Gao, Hui-Yuan; Meng, Qing-Wei

    2012-07-01

    Taking Rumex K-1 leaves as test materials, this paper studied the role of mitochondrial alternative oxidase (AOX) pathway in photoprotection under different light intensities. Under low light intensity (200 micromol x m(-2) x s(-1)), and after treated with salicylhydroxamic acid to inhibit the AOX pathway, the leaf actual photochemical efficiency of PS II, linear electron transport rate of photosynthesis, and photosynthetic O2 evolution rate all decreased significantly while the non-Q(B) reducing reaction center had a significant increase, indicating that under low light, the photoinhibition was aggravated while the scavenging enzymes of reactive oxygen species (ROS) increased, which avoided the over-accumulation of ROS and partially alleviated the photoinhibition of Rumex K-1 leaves. Under high light intensity (800 micromol x m(-2) x s(-1)), the inhibition of AOX pathway caused more severe photoinhibition, and the increased activities of ROS scavenging enzymes were insufficient to prevent the over-accumulation of ROS. This study demonstrated that AOX pathway played an important role in the photoprotection in Rumex K-1 leaves under both high and low light intensities, and the role of AOX pathway in photoprotection under high light could be irreplaceable by the other photoprotection pathways in chloroplast.

  20. Millimeter wave treatment induces apoptosis via activation of the mitochondrial-dependent pathway in human osteosarcoma cells.

    PubMed

    Wu, Guangwen; Chen, Xuzheng; Peng, Jun; Cai, Qiaoyan; Ye, Jinxia; Xu, Huifeng; Zheng, Chunsong; Li, Xihai; Ye, Hongzhi; Liu, Xianxiang

    2012-05-01

    Millimeter wave (MW) is an electromagnetic wave with a wavelength between 1 and 10 mm and a frequency of 30-300 GHz that causes multiple biological effects and has been used as a major component in physiotherapies for the clinical treatment of various types of diseases including cancers. However, the precise molecular mechanism of the anticancer activity of millimeter wave remains to be elucidated. In the present study, we investigated the cellular effects of the MW in the U-2OS human osteosarcoma cell line. Our results showed that MW induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner suggesting that MW inhibited the growth of U-2OS cells as demonstrated. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, MW treatment caused loss of plasma membrane asymmetry, release of cytochrome c, collapse of mitochondrial membrane potential, activation of caspase-9 and -3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of MW was due to mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of millimeter wave treatment.

  1. Estrogen receptor-β in mitochondria: implications for mitochondrial bioenergetics and tumorigenesis.

    PubMed

    Liao, Tien-Ling; Tzeng, Chii-Ruey; Yu, Chao-Lan; Wang, Yi-Pei; Kao, Shu-Huei

    2015-09-01

    Estrogen enhances mitochondrial function by enhancing mitochondrial biogenesis and sustaining mitochondrial energy-transducing capacity. Shifts in mitochondrial bioenergetic pathways from oxidative phosphorylation to glycolysis have been hypothesized to be involved in estrogen-induced tumorigenesis. Studies have shown that mitochondria are an important target of estrogen. Estrogen receptor-β (ERβ) has been shown to localize to mitochondria in a ligand-dependent or -independent manner and can affect mitochondrial bioenergetics and anti-apoptotic signaling. However, the functional role of mitochondrial ERβ in tumorigenesis remains unclear. Clinical studies of ERβ-related tumorigenesis have shown that ERβ stimulates mitochondrial metabolism to meet the high energy demands of processes such as cell proliferation, cell survival, and transformation. Thus, in elucidating the precise role of mitochondrial ERβ in cell transformation and tumorigenesis, it will be particularly valuable to explore new approaches for the development of medical treatments targeting mitochondrial ERβ-mediated mitochondrial function and preventing apoptosis.

  2. Inhibition of Drp1 by Mdivi-1 attenuates cerebral ischemic injury via inhibition of the mitochondria-dependent apoptotic pathway after cardiac arrest.

    PubMed

    Li, Y; Wang, P; Wei, J; Fan, R; Zuo, Y; Shi, M; Wu, H; Zhou, M; Lin, J; Wu, M; Fang, X; Huang, Z

    2015-12-17

    Mitochondrial fission is predominantly controlled by the activity of dynamin-related protein1 (Drp1), which has been reported to be involved in mitochondria apoptosis pathways. However, the role of Drp1 in a rat model of cardiac arrest remains unknown. In this study, we found that activation of Drp1 in the mitochondria was increased after cardiac arrest and inhibition of Drp1 by 1.2 mg/kg of mitochondrial division inhibitor-1 (Mdivi-1) administration after the restoration of spontaneous circulation (ROSC) significantly protected against cerebral ischemic injury, shown by the increased 72-h survival rate and improved neurological function. Moreover, the increase of the vital neuron and the reduction of cytochrome c (CytC) release, apoptosis-inducing factor (AIF) translocation and caspase-3 activation in the brain indicate that this protection might result from the suppression of neuron apoptosis. Altogether, these results indicated that Drp1 is activated after cardiac arrest and the inhibition of Drp1 is protective against cerebral ischemic injury in a rat of cardiac arrest model via inhibition of the mitochondrial apoptosis pathway.

  3. Interplay between lysosomal, mitochondrial and death receptor pathways during manganese-induced apoptosis in glial cells.

    PubMed

    Gorojod, R M; Alaimo, A; Porte Alcon, S; Saravia, F; Kotler, M L

    2017-04-04

    Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

  4. The MIA pathway: a key regulator of mitochondrial oxidative protein folding and biogenesis.

    PubMed

    Mordas, Amelia; Tokatlidis, Kostas

    2015-08-18

    Mitochondria are fundamental intracellular organelles with key roles in important cellular processes like energy production, Fe/S cluster biogenesis, and homeostasis of lipids and inorganic ions. Mitochondrial dysfunction is consequently linked to many human pathologies (cancer, diabetes, neurodegeneration, stroke) and apoptosis. Mitochondrial biogenesis relies on protein import as most mitochondrial proteins (about 10-15% of the human proteome) are imported after their synthesis in the cytosol. Over the last several years many mitochondrial translocation pathways have been discovered. Among them, the import pathway that targets proteins to the intermembrane space (IMS) stands out as it is the only one that couples import to folding and oxidation and results in the covalent modification of the incoming precursor that adopt internal disulfide bonds in the process (the MIA pathway). The discovery of this pathway represented a significant paradigm shift as it challenged the prevailing dogma that the endoplasmic reticulum is the only compartment of eukaryotic cells where oxidative folding can occur. The concept of the oxidative folding pathway was first proposed on the basis of folding and import data for the small Tim proteins that have conserved cysteine motifs and must adopt intramolecular disulfides after import so that they are retained in the organelle. The introduction of disulfides in the IMS is catalyzed by Mia40 that functions as a chaperone inducing their folding. The sulfhydryl oxidase Erv1 generates the disulfide pairs de novo using either molecular oxygen or, cytochrome c and other proteins as terminal electron acceptors that eventually link this folding process to respiration. The solution NMR structure of Mia40 (and supporting biochemical experiments) showed that Mia40 is a novel type of disulfide donor whose recognition capacity for its substrates relies on a hydrophobic binding cleft found adjacent to a thiol active CPC motif. Targeting of the

  5. The MIA Pathway: A Key Regulator of Mitochondrial Oxidative Protein Folding and Biogenesis

    PubMed Central

    2015-01-01

    Conspectus Mitochondria are fundamental intracellular organelles with key roles in important cellular processes like energy production, Fe/S cluster biogenesis, and homeostasis of lipids and inorganic ions. Mitochondrial dysfunction is consequently linked to many human pathologies (cancer, diabetes, neurodegeneration, stroke) and apoptosis. Mitochondrial biogenesis relies on protein import as most mitochondrial proteins (about 10–15% of the human proteome) are imported after their synthesis in the cytosol. Over the last several years many mitochondrial translocation pathways have been discovered. Among them, the import pathway that targets proteins to the intermembrane space (IMS) stands out as it is the only one that couples import to folding and oxidation and results in the covalent modification of the incoming precursor that adopt internal disulfide bonds in the process (the MIA pathway). The discovery of this pathway represented a significant paradigm shift as it challenged the prevailing dogma that the endoplasmic reticulum is the only compartment of eukaryotic cells where oxidative folding can occur. The concept of the oxidative folding pathway was first proposed on the basis of folding and import data for the small Tim proteins that have conserved cysteine motifs and must adopt intramolecular disulfides after import so that they are retained in the organelle. The introduction of disulfides in the IMS is catalyzed by Mia40 that functions as a chaperone inducing their folding. The sulfhydryl oxidase Erv1 generates the disulfide pairs de novo using either molecular oxygen or, cytochrome c and other proteins as terminal electron acceptors that eventually link this folding process to respiration. The solution NMR structure of Mia40 (and supporting biochemical experiments) showed that Mia40 is a novel type of disulfide donor whose recognition capacity for its substrates relies on a hydrophobic binding cleft found adjacent to a thiol active CPC motif. Targeting

  6. A3K2A3-induced apoptotic cell death of Leishmania amazonensis occurs through caspase- and ATP-dependent mitochondrial dysfunction.

    PubMed

    Garcia, Francielle Pelegrin; Henrique da Silva Rodrigues, Jean; Din, Zia Ud; Rodrigues-Filho, Edson; Ueda-Nakamura, Tânia; Auzély-Velty, Rachel; Nakamura, Celso Vataru

    2017-01-01

    Leishmaniasis is a neglected tropical disease that affects millions of people worldwide. Current therapies mainly rely on antimonial drugs that are inadequate because of their high toxicity and increased drug resistance. An urgent need exists to discover new, more effective, more affordable, and more target-specific drugs. Pathways that are associated with apoptosis-like cell death have been identified in unicellular eukaryotes, including protozoan parasites. In the present study, we studied the mechanism of cell death that is induced by A3K2A3 against L. amazonensis. A3K2A3 is a dibenzylideneacetone that has an acyclic dienone that is attached to aryl groups in both β-positions, which is similar to curcuminoids and chalcone structures. This compound was previously shown to be safe with regard to cytotoxicity and active against the parasite. Biochemical and morphological approaches were used in the present study. The results suggested that A3K2A3 caused mitochondrial dysfunction in L. amazonensis promastigotes, leading to mechanisms of cell death that share some common phenotypic features with metazoan apoptosis, such as an increase in reactive oxygen species production, a decrease in the adenosine triphosphate ratio, phosphatidylserine exposure, a decrease in cell volume, caspase production, and DNA fragmentation. Altogether, these findings indicate that apoptosis can indeed be triggered by chemotherapeutic agents.

  7. Modulation of mitochondrial capacity and angiogenesis by red wine polyphenols via estrogen receptor, NADPH oxidase and nitric oxide synthase pathways.

    PubMed

    Duluc, Lucie; Jacques, Caroline; Soleti, Raffaella; Iacobazzi, Francesco; Simard, Gilles; Andriantsitohaina, Ramaroson

    2013-04-01

    Red wine polyphenolic compounds (RWPC) are reported to exert vasculoprotective properties on endothelial cells, involving nitric oxide (NO) release via a redox-sensitive pathway. This NO release involves the activation of the estrogen receptor-alpha (ERα). Paradoxical effects of a RWPC treatment occur in a rat model of post-ischemic neovascularization, where a low-dose is pro-angiogenic while a higher dose is anti-angiogenic. NO and ERα are key regulators of mitochondrial capacity, and angiogenesis is a highly energetic process associated with mitochondrial biogenesis. However, whether RWPC induces changes in mitochondrial capacity has never been addressed. We investigated the effects of RWPC at low (10(-4)g/l, LCP) and high concentration (10(-2)g/l, HCP) in human endothelial cells. Mitochondrial respiration, expression of mitochondrial biogenesis factors and mitochondrial DNA content were assessed using oxygraphy and quantitative PCR respectively. In vitro capillary formation using ECM gel(®) was also performed. Treatment with LCP increased mitochondrial respiration, with a maximal effect achieved at 48h. LCP also increased expression of several mitochondrial biogenesis factors and mitochondrial DNA content. In contrast, HCP did not affect these parameters. Furthermore, LCP modulated both mitochondrial capacity and angiogenesis through mechanisms sensitive to ER, NADPH oxidase and NO-synthase inhibitors. Finally, the inhibition of mitochondrial protein synthesis abolished the pro-angiogenic capacity of LCP. These results suggest a possible association between the modulation of mitochondrial capacity by LCP and its pro-angiogenic activity. These data provide evidence for a role of mitochondria in the regulation of angiogenesis by RWPC.

  8. Resveratrol prevents doxorubicin cardiotoxicity through mitochondrial stabilization and the Sirt1 pathway.

    PubMed

    Danz, Elizabeth D Brookins; Skramsted, Jeremy; Henry, Nicholas; Bennett, James A; Keller, Rebecca S

    2009-06-15

    Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, its incidence of cardiotoxicity compromises its therapeutic index. DOX-induced heart failure is thought to be caused by reduction/oxidation cycling of DOX to generate oxidative stress and cardiomyocyte cell death. Resveratrol (RV), a stilbene found in red wine, has been reported to play a cardioprotective role in diseases associated with oxidative stress. The objective of this study was to test the ability of RV to protect against DOX-induced cardiomyocyte death. We hypothesized that RV protects cardiomyocytes from DOX-induced oxidative stress and subsequent cell death through changes in mitochondrial function. DOX induced a rapid increase in reactive oxygen species (ROS) production in cardiac cell mitochondria, which was inhibited by pretreatment with RV, most likely owing to an increase in MnSOD activity. This effect of RV caused additional polarization of the mitochondria in the absence and presence of DOX to increase mitochondrial function. RV pretreatment also prevented DOX-induced cardiomyocyte death. The protective ability of RV against DOX was abolished when Sirt1 was inhibited by nicotinamide. Our data suggest that RV protects against DOX-induced oxidative stress through changes in mitochondrial function, specifically the Sirt1 pathway leading to cardiac cell survival.

  9. Centella asiatica modulates antioxidant and mitochondrial pathways and improves cognitive function in mice

    PubMed Central

    Gray, Nora E.; Harris, Christopher J.; Quinn, Joseph F.; Soumyanath, Amala

    2016-01-01

    Ethnopharmacological relevance This study investigates the cognitive enhancing effects of the plant Centella asiatica which is widely used Ayurvedic and traditional Chinese medicine. Aim of the study The goal of this study was to determine the effects of a water extract of the medicinal plant Centella asiatica (CAW) on cognitive ability as well as mitochondrial and antioxidant response pathways in vivo. Materials and methods Old and young C57BL/6 mice were treated with CAW (2mg/mL) in their drinking water. Learning and memory was assessed using Morris Water Maze (MWM) and then tissue was collected and gene expression analyzed. Results CAW improved performance in the MWM in aged animals and had a modest effect on the performance of young animals. CAW also increased the expression of mitochondrial and antioxidant response genes in the brain and liver of both young and old animals. Expression of synaptic markers was also increased in the hippocampus and frontal cortex, but not in the cerebellum of CAW-treated animals. Conclusions These data indicate a cognitive enhancing effect of CAW in healthy mice. The gene expression changes caused by CAW suggest a possible effect on mitochondrial biogenesis, which in conjunction with activation of antioxidant response genes could contribute to cognitive improvement. PMID:26785167

  10. Cardiac dysfunction, mitochondrial architecture, energy production, and inflammatory pathways: Interrelated aspects in endotoxemia and sepsis.

    PubMed

    Alvarez, Silvia; Vico, Tamara; Vanasco, Virginia

    2016-12-01

    Septic patients with myocardial dysfunction have a 3-fold increase in mortality compared with patients without cardiovascular impairment, and usually show myocarditis, disruption of the contractile apparatus, increased amounts of interstitial collagen, and damaged mitochondria. The presence of nitric oxide and cytokines in cardiac tissue constitute the molecular markers and the intracellular messengers of inflammatory conditions in the heart due to the onset of sepsis and endotoxemia, derived from the nuclear factor-κB pathway activation and proinflammatory gene transcription. Sepsis occurs with an exacerbated inflammatory response that damages tissue mitochondria and impaired bioenergetic processes. The heart consumes 20-30 times its own weight in adenosine triphosphate every day, and 90% of this molecule is derived from mitochondrial oxidative phosphorylation. Cardiac energy management is comprised in sepsis and endotoxemia; both a deficit in energy production and alterations in the source of energy substrates are believed to be involved in impaired cardiac function. Although several hypotheses try to explain the molecular mechanisms underlying the complex condition of sepsis and endotoxemia, the current view is that these syndromes are the result of an intricate balance between prevailing levels of mitochondrial stress, biogenesis/autophagy signaling and mitochondria quality control processes, rather on a single factor. The aim of this review is to discuss current hypothesis of cardiac dysfunction related to energy metabolism and mitochondrial function in experimental models of sepsis and endotoxemia, and to introduce the importance of lipids (mainly cardiolipin) in the mechanism of cardiac energy mismanagement in these inflammatory conditions.

  11. A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4.

    PubMed

    Evstafieva, A G; Garaeva, A A; Khutornenko, A A; Klepikova, A V; Logacheva, M D; Penin, A A; Novakovsky, G E; Kovaleva, I E; Chumakov, P M

    2014-11-06

    Generation of energy in mitochondria is subjected to physiological regulation at many levels, and its malfunction may result in mitochondrial diseases. Mitochondrial dysfunction is associated with different environmental influences or certain genetic conditions, and can be artificially induced by inhibitors acting at different steps of the mitochondrial electron transport chain (ETC). We found that a short-term (5 h) inhibition of ETC complex III with myxothiazol results in the phosphorylation of translation initiation factor eIF2α and upregulation of mRNA for the activating transcription factor 4 (ATF4) and several ATF4-regulated genes. The changes are characteristic for the adaptive integrated stress response (ISR), which is known to be triggered by unfolded proteins, nutrient and metabolic deficiency, and mitochondrial dysfunctions. However, after a prolonged incubation with myxothiazol (13-17 h), levels of ATF4 mRNA and ATF4-regulated transcripts were found substantially suppressed. The suppression was dependent on the p53 response, which is triggered by the impairment of the complex III-dependent de novo biosynthesis of pyrimidines by mitochondrial dihydroorotate dehydrogenase. The initial adaptive induction of ATF4/ISR acted to promote viability of cells by attenuating apoptosis. In contrast, the induction of p53 upon a sustained inhibition of ETC complex III produced a pro-apoptotic effect, which was additionally stimulated by the p53-mediated abrogation of the pro-survival activities of the ISR. Interestingly, a sustained inhibition of ETC complex I by piericidine did not induce the p53 response and stably maintained the pro-survival activation of ATF4/ISR. We conclude that a downregulation of mitochondrial ETC generally induces adaptive pro-survival responses, which are specifically abrogated by the suicidal p53 response triggered by the genetic risks of the pyrimidine nucleotide deficiency.

  12. Transaldolase deficiency influences the pentose phosphate pathway, mitochondrial homoeostasis and apoptosis signal processing.

    PubMed

    Qian, Yueming; Banerjee, Sanjay; Grossman, Craig E; Amidon, Wendy; Nagy, Gyorgy; Barcza, Maureen; Niland, Brian; Karp, David R; Middleton, Frank A; Banki, Katalin; Perl, Andras

    2008-10-01

    TAL (transaldolase) was originally described in the yeast as an enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall reason for its existence is unclear. Recently, deletion of Ser(171) (TALDeltaS171) was found in five patients causing inactivation, proteasome-mediated degradation and complete deficiency of TAL. In the present study, microarray and follow-up Western-blot, enzyme-activity and metabolic studies of TALDeltaS171 TD (TAL-deficient) lymphoblasts revealed co-ordinated changes in the expression of genes involved in the PPP, mitochondrial biogenesis, oxidative stress, and Ca(2+) fluxing. Sedoheptulose 7-phosphate was accumulated, whereas G6P (glucose 6-phosphate) was depleted, indicating a failure to recycle G6P for the oxidative branch of the PPP. Nucleotide analysis showed depletion of NADPH and NAD(+) and accumulation of ADP-ribose. TD cells have diminished Deltapsi(m) (mitochondrial transmembrane potential) and increased mitochondrial mass associated with increased production of nitric oxide and ATP. TAL deficiency resulted in enhanced spontaneous and H(2)O(2)-induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses NAD(+) into ADP-ribose, a trigger of Ca(2+) release from the endoplasmic reticulum that, in turn, facilitated CD20-induced apoptosis. By contrast, TD cells were resistant to CD95/Fas-induced apoptosis, owing to a dependence of caspase activity on redox-sensitive cysteine residues. Normalization of TAL activity by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca(2+) levels, suppressed H(2)O(2)- and CD20-induced apoptosis and enhanced Fas-induced cell death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca(2+) fluxing and apoptosis.

  13. Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells.

    PubMed

    An, Hyun-Kyu; Kim, Kyoung-Sook; Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.

  14. Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

    PubMed Central

    Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy. PMID:25490748

  15. DPI induces mitochondrial superoxide-mediated apoptosis.

    PubMed

    Li, Nianyu; Ragheb, Kathy; Lawler, Gretchen; Sturgis, Jennie; Rajwa, Bartek; Melendez, J Andres; Robinson, J Paul

    2003-02-15

    The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.

  16. Response of mitochondrial antioxidant system and respiratory pathways to reactive nitrogen species in pea leaves.

    PubMed

    Martí, María C; Florez-Sarasa, Igor; Camejo, Daymi; Pallol, Beatriz; Ortiz, Ana; Ribas-Carbó, Miquel; Jiménez, Ana; Sevilla, Francisca

    2013-02-01

    Nitric oxide (NO) has emerged as an important signaling molecule in plants, but little is known about the effects of reactive nitrogen species in plant mitochondria. In this study, the effects of DETA-NONOate, a pure NO slow generator, and of SIN-1 (3-morpholinosydnonimine), a peroxynitrite producer, on the activities of respiratory pathways, enzymatic and non-enzymatic antioxidants have been investigated in isolated mitochondria from pea leaves. No significant changes in lipid peroxidation, protein oxidation or in ascorbate and glutathione redox state were observed after DETA-NONOate treatments whereas cytochrome pathway (CP) respiration was reversibly inhibited and alternative pathway (AP) respiration showed little inhibition. On the other hand, NO did not affect neither activities of Mn superoxide dismutase (Mn-SOD) nor enzymes involved in the ascorbate and glutathione regeneration in mitochondria except for ascorbate peroxidase (APX), which was reversely inhibited depending on ascorbate concentration. Finally, SIN-1 treatment of mitochondria produced a decrease in CP respiration, an increase in protein oxidation and strongly inhibited APX activity (90%), with glutathione reductase and dehydroascorbate reductase (DHAR) being moderately inhibited (30 and 20%, respectively). This treatment did not affect monodehydroascorbate reductase (MDHAR) and Mn-SOD activities. Results showed that mitochondrial nitrosative stress was not necessarily accompanied by oxidative stress. We suggest that NO-resistant AP and mitochondrial APX may be important components of the H(2) O(2) -signaling pathways under nitrosative stress induced by NO in this organelle. Also, MDHAR and DHAR, via ascorbate regeneration, could constitute an essential antioxidant defense together with Mn-SOD, against NO and ONOO(-) stress in plant mitochondria.

  17. DUX4-induced dsRNA and MYC mRNA stabilization activate apoptotic pathways in human cell models of facioscapulohumeral dystrophy.

    PubMed

    Shadle, Sean C; Zhong, Jun Wen; Campbell, Amy E; Conerly, Melissa L; Jagannathan, Sujatha; Wong, Chao-Jen; Morello, Timothy D; van der Maarel, Silvère M; Tapscott, Stephen J

    2017-03-01

    Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of DUX4 in skeletal muscle cells. DUX4 is a transcription factor that activates genes normally associated with stem cell biology and its mis-expression in FSHD cells results in apoptosis. To identify genes and pathways necessary for DUX4-mediated apoptosis, we performed an siRNA screen in an RD rhabdomyosarcoma cell line with an inducible DUX4 transgene. Our screen identified components of the MYC-mediated apoptotic pathway and the double-stranded RNA (dsRNA) innate immune response pathway as mediators of DUX4-induced apoptosis. Further investigation revealed that DUX4 expression led to increased MYC mRNA, accumulation of nuclear dsRNA foci, and activation of the dsRNA response pathway in both RD cells and human myoblasts. Nuclear dsRNA foci were associated with aggregation of the exon junction complex component EIF4A3. The elevation of MYC mRNA, dsRNA accumulation, and EIF4A3 nuclear aggregates in FSHD muscle cells suggest that these processes might contribute to FSHD pathophysiology.

  18. DUX4-induced dsRNA and MYC mRNA stabilization activate apoptotic pathways in human cell models of facioscapulohumeral dystrophy

    PubMed Central

    Shadle, Sean C.; Jagannathan, Sujatha; Wong, Chao-Jen; Morello, Timothy D.; van der Maarel, Silvère M.

    2017-01-01

    Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of DUX4 in skeletal muscle cells. DUX4 is a transcription factor that activates genes normally associated with stem cell biology and its mis-expression in FSHD cells results in apoptosis. To identify genes and pathways necessary for DUX4-mediated apoptosis, we performed an siRNA screen in an RD rhabdomyosarcoma cell line with an inducible DUX4 transgene. Our screen identified components of the MYC-mediated apoptotic pathway and the double-stranded RNA (dsRNA) innate immune response pathway as mediators of DUX4-induced apoptosis. Further investigation revealed that DUX4 expression led to increased MYC mRNA, accumulation of nuclear dsRNA foci, and activation of the dsRNA response pathway in both RD cells and human myoblasts. Nuclear dsRNA foci were associated with aggregation of the exon junction complex component EIF4A3. The elevation of MYC mRNA, dsRNA accumulation, and EIF4A3 nuclear aggregates in FSHD muscle cells suggest that these processes might contribute to FSHD pathophysiology. PMID:28273136

  19. Genes and Pathways Involved in Adult Onset Disorders Featuring Muscle Mitochondrial DNA Instability

    PubMed Central

    Ahmed, Naghia; Ronchi, Dario; Comi, Giacomo Pietro

    2015-01-01

    Replication and maintenance of mtDNA entirely relies on a set of proteins encoded by the nuclear genome, which include members of the core replicative machinery, proteins involved in the homeostasis of mitochondrial dNTPs pools or deputed to the control of mitochondrial dynamics and morphology. Mutations in their coding genes have been observed in familial and sporadic forms of pediatric and adult-onset clinical phenotypes featuring mtDNA instability. The list of defects involved in these disorders has recently expanded, including mutations in the exo-/endo-nuclease flap-processing proteins MGME1 and DNA2, supporting the notion that an enzymatic DNA repair system actively takes place in mitochondria. The results obtained in the last few years acknowledge the contribution of next-generation sequencing methods in the identification of new disease loci in small groups of patients and even single probands. Although heterogeneous, these genes can be conveniently classified according to the pathway to which they belong. The definition of the molecular and biochemical features of these pathways might be helpful for fundamental knowledge of these disorders, to accelerate genetic diagnosis of patients and the development of rational therapies. In this review, we discuss the molecular findings disclosed in adult patients with muscle pathology hallmarked by mtDNA instability. PMID:26251896

  20. Panax notoginseng saponins attenuates cisplatin-induced nephrotoxicity via inhibiting the mitochondrial pathway of apoptosis.

    PubMed

    Liu, Xinwen; Huang, Zhenguang; Zou, Xiaoqin; Yang, Yufang; Qiu, Yue; Wen, Yan

    2014-01-01

    The goal of this experiment was to investigate the protective effect and the molecular mechanism of Panax Notoginseng Saponins (PNS) on cisplatin-induced nephrotoxicity through mitochondrial pathway of apoptosis. The rats underwent intraperitoneal injection with a single dose of cisplatin, a subset of rats were also intraperitoneally injected with 31.35 mg/kg PNS once a day for 8 days. At day 1, 4 and 8 after exposure to cisplatin, the concentrations of blood urea nitrogen (BUN), serum creatinine (Scr) and urinary N-acetyl-β-D-Glucosaminidase (NAG) were determined using commercial kits. The pathological change of renal tissue were examined using H & E staining and transmission electron microscopy. The rate of apoptosis and the expression of Bcl-2 in rat renal tissue were detected by using TUNEL staining and Western bloting, respectively. And the expressions of Bax and caspases 9 were detected by immunnohistochemistry. The results showed that PNS significantly protected against cisplatin-induced nephrotoxicity, as evidenced by the decrease in concentration of blood BUN, Scr and urinary NAG, as well as the attenuation of renal histopathological damage. Furthermore, PNS reduced the rate of apoptosis, and the mechanism studies showed that PNS inhibited the expression of Bax and caspase 9, while increased the expression of Bcl-2. This study first demonstrated that PNS can protect against cisplatin-induced nephrotoxicity and reduce renal tissue apoptosis via inhibiting the mitochondrial pathway.

  1. Curcumin attenuates Mancozeb-induced toxicity in rat thymocytes through mitochondrial survival pathway.

    PubMed

    Pavlovic, Voja; Cekic, Snezana; Ciric, Milan; Krtinic, Dane; Jovanovic, Jelena

    2016-02-01

    The widely used fungicide Mancozeb (Man) has been shown to cause genotoxic effects in rodents and toxicological manifestations in different cells, mainly by altering the antioxidant defense in cells. On the other hand, curcumin (Cur), a natural phenolic compound, is thought to possess anti-inflammatory and antioxidant properties. Here, we investigated the possible protective role of Cur on Man-induced toxicity in rat thymocytes and potential mechanism involved. Rat thymocytes were treated with Man(0.01 μg/ml) and/or increasing Cur(0.3, 1, 3 μM) concentrations and levels of cell viability, apoptosis, mitochondrial membrane potential (MMP),Bcl-2, Bax protein expression, caspase-3 and -9 activity and p38 MAPK signaling involvement were examined. Cells treated with Man displayed increased cell toxicity, hypodiploid cells, caspase-3 and -9 activity, Bax protein expression, followed with decreased MMP and Bcl-2 protein expression. Inhibition of p38 MAPK signaling pathway markedly reduced apoptosis rate and caspase-3 activity in thymocytes exposed to Man. Application of increasing Cur (1, 3 μM) concentrations resulted with significantly reduced cytotoxicity, apoptosis, caspase-3, -9 activity, Bax protein expression, together with increased MMP and Bcl-2 protein expression in rat thymocytes. These result suggest that certain Cur concentrations may mediate Man-induced rat thymocytes toxicity through mitochondrial survival pathway, which may be useful in preventing possible secondary immunological consequences induced by Man.

  2. Genes and Pathways Involved in Adult Onset Disorders Featuring Muscle Mitochondrial DNA Instability.

    PubMed

    Ahmed, Naghia; Ronchi, Dario; Comi, Giacomo Pietro

    2015-08-05

    Replication and maintenance of mtDNA entirely relies on a set of proteins encoded by the nuclear genome, which include members of the core replicative machinery, proteins involved in the homeostasis of mitochondrial dNTPs pools or deputed to the control of mitochondrial dynamics and morphology. Mutations in their coding genes have been observed in familial and sporadic forms of pediatric and adult-onset clinical phenotypes featuring mtDNA instability. The list of defects involved in these disorders has recently expanded, including mutations in the exo-/endo-nuclease flap-processing proteins MGME1 and DNA2, supporting the notion that an enzymatic DNA repair system actively takes place in mitochondria. The results obtained in the last few years acknowledge the contribution of next-generation sequencing methods in the identification of new disease loci in small groups of patients and even single probands. Although heterogeneous, these genes can be conveniently classified according to the pathway to which they belong. The definition of the molecular and biochemical features of these pathways might be helpful for fundamental knowledge of these disorders, to accelerate genetic diagnosis of patients and the development of rational therapies. In this review, we discuss the molecular findings disclosed in adult patients with muscle pathology hallmarked by mtDNA instability.

  3. Bmi1 regulates mitochondrial function and the DNA damage response pathway.

    PubMed

    Liu, Jie; Cao, Liu; Chen, Jichun; Song, Shiwei; Lee, In Hye; Quijano, Celia; Liu, Hongjun; Keyvanfar, Keyvan; Chen, Haoqian; Cao, Long-Yue; Ahn, Bong-Hyun; Kumar, Neil G; Rovira, Ilsa I; Xu, Xiao-Ling; van Lohuizen, Maarten; Motoyama, Noboru; Deng, Chu-Xia; Finkel, Toren

    2009-05-21

    Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1(-/-) phenotype. Here we demonstrate that cells derived from Bmi1(-/-) mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1(-/-) mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.

  4. An anti-apoptotic viral protein that recruits Bax to mitochondria.

    PubMed

    Poncet, Delphine; Larochette, Nathanael; Pauleau, Anne-Laure; Boya, Patricia; Jalil, Abdel-Ali; Cartron, Pierre-Francois; Vallette, Francois; Schnebelen, Céline; Bartle, Laura M; Skaletskaya, Anna; Boutolleau, David; Martinou, Jean-Claude; Goldmacher, Victor S; Kroemer, Guido; Zamzami, Naoufal

    2004-05-21

    The viral mitochondria-localized inhibitor of apoptosis (vMIA), encoded by the UL37 gene of human cytomegalovirus, inhibits apoptosis-associated mitochondrial membrane permeabilization by a mechanism different from that of Bcl-2. Here we show that vMIA induces several changes in Bax that resemble those found in apoptotic cells yet take place in unstimulated, non-apoptotic vMIA-expressing cells. These changes include the constitutive localization of Bax at mitochondria, where it associates tightly with the mitochondrial membrane, forming high molecular weight aggregates that contain vMIA. vMIA recruits Bax to mitochondria but delays relocation of caspase-8-activated truncated Bid-green fluorescent protein (GFP) (t-Bid-GFP) to mitochondria. The ability of vMIA and its deletion mutants to associate with Bax and to induce relocation of Bax to mitochondria correlates with their anti-apoptotic activity and with their ability to suppress mitochondrial membrane permeabilization. Taken together, our data indicate that vMIA blocks apoptosis via its interaction with Bax. vMIA neutralizes Bax by recruiting it to mitochondria and "freezing" its pro-apoptotic activity. These data unravel a novel strategy of subverting an intrinsic pathway of apoptotic signaling.

  5. Alteration in inflammatory/apoptotic pathway and histone modifications by nordihydroguaiaretic acid prevents acute pancreatitis in swiss albino mice.

    PubMed

    Mahajan, Ujwal Mukund; Gupta, Chanchal; Wagh, Preshit Ravindra; Karpe, Pinakin Arun; Tikoo, Kulbhushan

    2011-11-01

    Reactive oxygen radicals, pro-inflammatory mediators and cytokines have been implicated in caerulein induced acute pancreatitis. Nordihydroguaiaretic acid (NDGA), a plant lignin, has marked anti-inflammatory properties. The present study aimed to investigate the possible protective effect of NDGA against caerulein induced pancreatitis. Acute pancreatitis was induced by intraperitoneal administration of eight doses of caerulein in male swiss albino mice. NDGA was administered after 9 h of acute pancreatitis induction. Pancreatic damage and the protective effect of NDGA were assessed by oxidative stress parameters and histopathology of pancreas. The mRNA expression of heat shock proteins (DNAJ C15 and HSPD1) was examined by real-time RT-PCR analysis. Expression of HSP 27, NF-κB, TNF-α, p-p38, Bcl-2, p-PP2A, procaspase-3, caspase-3 and histone modifications were examined by western blotting. NDGA attenuated the oxidative stress, led to increased plasma α-amylase and decreased IGF-1 in AP mice. It modulated the mRNA and protein levels of heat shock proteins and reduced the expression of NF-κB, TNF-α and p-p38. It increased the number of TUNEL positive apoptotic cells in the pancreas of AP mice. In addition, NDGA prevented the changes in modifications of histone H3 in acute pancreatitis. To best of our knowledge, this is the first report which suggests that NDGA prevents the progression of acute pancreatitis by involving alteration of histone H3 modifications and modulating the expression of genes involved in inflammatory/apoptotic cascade, which may be responsible for decreased necrosis and increased apoptosis in this model of acute pancreatitis.

  6. Multiple alphaII-spectrin breakdown products distinguish calpain and caspase dominated necrotic and apoptotic cell death pathways.

    PubMed

    Zhang, Zhiqun; Larner, Stephen F; Liu, Ming Cheng; Zheng, Wenrong; Hayes, Ronald L; Wang, Kevin K W

    2009-11-01

    Apoptosis and oncotic necrosis in neuronal and glial cells have been documented in many neurological diseases. Distinguishing between these two major types of cell death in different neurological diseases is needed in order to better reveal the injury mechanisms so as to open up opportunities for therapy development. Accumulating evidence suggests apoptosis and oncosis epitomize the extreme ends of a broad spectrum of morphological and biochemical events. Biochemical markers that can distinguish between the calpain and caspase dominated types of cell death would help in this process. In this study, three chemical agents, maitotoxin (MTX), staurosporine (STS) and thylenediaminetetraacetic acid (EDTA), were used to induce different types of cell death in PC12 neuronal-like cells. MTX-induced necrosis, as determined by the increased levels of calpain-specific cleaved fragments of spectrin by antibodies specific to the calpain-cleaved 150 kDa alphaII-spectrin breakdown product (SBDP150) and 145 kDa alphaII-spectrin breakdown product (SBDP145). In this paradigm, there were no detectable SBDP150i and SBDP120 fragments as determined by antibodies specific to the caspase-cleaved specific fragments similar to those seen in the EDTA-mediated apoptotic PC-12 cells. In contrast to the calpain specific MTX necrosis treatment and the caspase EDTA apoptotic treatment is the STS treatment which induced both proteases as shown by the increase in all the SBDP fragments. Furthermore, compared to SBDP150, SBDP145 appears to be a more specific and sensitive biomarker for calpain activation. Taken together, our results suggested calpains and caspases which dominate the two major types of cell death could be independently discriminated by specifically examining the multiple alphaII-spectrin cleavage breakdown products.

  7. Redox Regulation of Mitochondrial Function

    PubMed Central

    Handy, Diane E.

    2012-01-01

    Abstract Redox-dependent processes influence most cellular functions, such as differentiation, proliferation, and apoptosis. Mitochondria are at the center of these processes, as mitochondria both generate reactive oxygen species (ROS) that drive redox-sensitive events and respond to ROS-mediated changes in the cellular redox state. In this review, we examine the regulation of cellular ROS, their modes of production and removal, and the redox-sensitive targets that are modified by their flux. In particular, we focus on the actions of redox-sensitive targets that alter mitochondrial function and the role of these redox modifications on metabolism, mitochondrial biogenesis, receptor-mediated signaling, and apoptotic pathways. We also consider the role of mitochondria in modulating these pathways, and discuss how redox-dependent events may contribute to pathobiology by altering mitochondrial function. Antioxid. Redox Signal. 16, 1323–1367. PMID:22146081

  8. De-novo NAD+ synthesis regulates SIRT1-FOXO1 apoptotic pathway in response to NQO1 substrates in lung cancer cells

    PubMed Central

    Cheng, Xuefang; Li, Qingran; Liu, Fang; Ye, Hui; Zhao, Min; Wang, Hong; Wang, Guangji; Hao, Haiping

    2016-01-01

    Tryptophan metabolism is essential in diverse kinds of tumors via regulating tumor immunology. However, the direct role of tryptophan metabolism and its signaling pathway in cancer cells remain largely elusive. Here, we establish a mechanistic link from L-type amino acid transporter 1 (LAT1) mediated transport of tryptophan and the subsequent de-novo NAD+ synthesis to SIRT1-FOXO1 regulated apoptotic signaling in A549 cells in response to NQO1 activation. In response to NQO1 activation, SIRT1 is repressed leading to the increased cellular accumulation of acetylated FOXO1 that transcriptionally activates apoptotic signaling. Decreased uptake of tryptophan due to the downregulation of LAT1 coordinates with PARP-1 hyperactivation to induce rapid depletion of NAD+ pool. Particularly, the LAT1-NAD+-SIRT1 signaling is activated in tumor tissues of patients with non-small cell lung cancer. Because NQO1 activation is characterized with oxidative challenge induced DNA damage, these results suggest that LAT1 and de-novo NAD+ synthesis in NSCLC cells may play essential roles in sensing excessive oxidative stress. PMID:27566573

  9. Amelioration of nandrolone decanoate-induced testicular and sperm toxicity in rats by taurine: Effects on steroidogenesis, redox and inflammatory cascades, and intrinsic apoptotic pathway

    SciTech Connect

    Ahmed, Maha A.E.

    2015-02-01

    The wide abuse of the anabolic steroid nandrolone decanoate by athletes and adolescents for enhancement of sporting performance and physical appearance may be associated with testicular toxicity and infertility. On the other hand, taurine; a free β-amino acid with remarkable antioxidant activity, is used in taurine-enriched beverages to boost the muscular power of athletes. Therefore, the purpose of this study was to investigate the mechanisms of the possible protective effects of taurine on nandrolone decanoate-induced testicular and sperm toxicity in rats. To achieve this aim, male Wistar rats were randomly distributed into four groups and administered either vehicle, nandrolone decanoate (10 mg/kg/week, I.M.), taurine (100 mg/kg/day, p.o.) or combination of taurine and nandrolone decanoate, for 8 successive weeks. Results of the present study showed that taurine reversed nandrolone decanoate-induced perturbations in sperm characteristics, normalized serum testosterone level, and restored the activities of the key steroidogenic enzymes; 3β-HSD, and 17β-HSD. Moreover, taurine prevented nandrolone decanoate-induced testicular toxicity and DNA damage by virtue of its antioxidant, anti-inflammatory, and anti-apoptotic effects. This was evidenced by taurine-induced modulation of testicular LDH-x activity, redox markers (MDA, NO, GSH contents, and SOD activity), inflammatory indices (TNF-α, ICAM-1 levels, and MMP-9 gene expression), intrinsic apoptotic pathway (cytochrome c gene expression and caspase-3 content), and oxidative DNA damage markers (8-OHdG level and comet assay). In conclusion, at the biochemical and histological levels, taurine attenuated nandrolone decanoate-induced poor sperm quality and testicular toxicity in rats. - Highlights: • Nandrolone decanoate (ND) disrupts sperm profile and steroidogenesis in rats. • ND upregulates gene expression of inflammatory and apoptotic markers. • Taurine normalizes sperm profile and serum testosterone level

  10. Activation of the mitochondrial signaling pathway in response to organic solvent stress in yeast.

    PubMed

    Nishida-Aoki, Nao; Mori, Hitoshi; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-05-01

    In Saccharomyces cerevisiae, we have demonstrated that organic solvent stress activated the pleiotropic drug resistance (PDR) pathway, which involves the transcription factors Pdr1p and Pdr3p. Pdr1p and Pdr3p are functionally homologous in multidrug resistance, although Pdr3p has been reported to have some distinct functions. Here, we analyzed the functions of Pdr1p and Pdr3p during the cellular response to isooctane, as a representative of organic solvents, and observed the differential functions of Pdr1p and Pdr3p. In response to organic solvent stress, only Pdr3p contributed to the regulation of downstream genes of the PDR pathway, while Pdr1p had a rather inhibitory role on transcriptional induction through competition with Pdr3p for binding to their recognition sequence, pleiotropic drug response element. Our results demonstrated that organic solvent stress was likely to damage mitochondria, causing generation of reactive oxygen species and mitochondrial fragmentation, and to activate retrograde signaling pathway via Pdr3p to upregulate PDR5 expression. Therefore, the unique function of Pdr3p in organic solvent stress distinguishes this pathway from the multidrug response.

  11. BCL-2 Antagonism to Target the Intrinsic Mitochondrial Pathway of Apoptosis.

    PubMed

    Gibson, Christopher J; Davids, Matthew S

    2015-11-15

    Despite significant improvements in treatment, cure rates for many cancers remain suboptimal. The rise of cytotoxic chemotherapy has led to curative therapy for a subset of cancers, though intrinsic treatment resistance is difficult to predict for individual patients. The recent wave of molecularly targeted therapies has focused on druggable-activating mutations, and is thus limited to specific subsets of patients. The lessons learned from these two disparate approaches suggest the need for therapies that borrow aspects of both, targeting biologic properties of cancer that are at once distinct from normal cells and yet common enough to make the drugs widely applicable across a range of cancer subtypes. The intrinsic mitochondrial pathway of apoptosis represents one such promising target for new therapies, and successfully targeting this pathway has the potential to alter the therapeutic landscape of therapy for a variety of cancers. Here, we discuss the biology of the intrinsic pathway of apoptosis, an assay known as BH3 profiling that can interrogate this pathway, early attempts to target BCL-2 clinically, and the recent promising results with the BCL-2 antagonist venetoclax (ABT-199) in clinical trials in hematologic malignancies. See all articles in this CCR Focus section, "Cell Death and Cancer Therapy."

  12. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells

    PubMed Central

    Reyes-Zurita, Fernando J.; Rufino-Palomares, Eva E.; García-Salguero, Leticia; Peragón, Juan; Medina, Pedro P.; Parra, Andrés; Cascante, Marta; Lupiáñez, José A.

    2016-01-01

    Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin. PMID:26751572

  13. Association of Genes, Pathways, and Haplogroups of the Mitochondrial Genome with the Risk of Colorectal Cancer: The Multiethnic Cohort.

    PubMed

    Li, Yuqing; Beckman, Kenneth B; Caberto, Christian; Kazma, Remi; Lum-Jones, Annette; Haiman, Christopher A; Le Marchand, Loïc; Stram, Daniel O; Saxena, Richa; Cheng, Iona

    2015-01-01

    The mitochondrial genome encodes for the synthesis of 13 proteins that are essential for the oxidative phosphorylation (OXPHOS) system. Inherited variation in mitochondrial genes may influence cancer development through changes in mitochondrial proteins, altering the OXPHOS process, and promoting the production of reactive oxidative species. To investigate the role of the OXPHOS pathway and mitochondrial genes in colorectal cancer (CRC) risk, we tested 185 mitochondrial SNPs (mtSNPs), located in 13 genes that comprise four complexes of the OXPHOS pathway and mtSNP groupings for rRNA and tRNA, in 2,453 colorectal cancer cases and 11,930 controls from the Multiethnic Cohort Study. Using the sequence kernel association test, we examined the collective set of 185 mtSNPs, as well as subsets of mtSNPs grouped by mitochondrial pathways, complexes, and genes, adjusting for age, sex, principal components of global ancestry, and self-reported maternal race/ethnicity. We also tested for haplogroup associations using unconditional logistic regression, adjusting for the same covariates. Stratified analyses were conducted by self-reported maternal race/ethnicity. In European Americans, a global test of all genetic variants of the mitochondrial genome identified an association with CRC risk (P = 0.04). In mtSNP-subset analysis, the NADH dehydrogenase 2 (MT-ND2) gene in Complex I was associated with CRC risk at a P-value of 0.001 (q = 0.015). In addition, haplogroup T was associated with CRC risk (OR = 1.66, 95% CI: 1.19-2.33, P = 0.003). No significant mitochondrial pathway and gene associations were observed in the remaining four racial/ethnic groups--African Americans, Asian Americans, Latinos, and Native Hawaiians. In summary, our findings suggest that variations in the mitochondrial genome and particularly in the MT-ND2 gene may play a role in CRC risk among European Americans, but not in other maternal racial/ethnic groups. Further replication is warranted and future studies

  14. Carboxylation of multiwalled carbon nanotube attenuated the cytotoxicity by limiting the oxidative stress initiated cell membrane integrity damage, cell cycle arrestment, and death receptor mediated apoptotic pathway.

    PubMed

    Liu, Zhenbao; Liu, Yanfei; Peng, Dongming

    2015-08-01

    In this study, the effects of carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) on human normal liver cell line L02 was compared with that of pristine multiwalled carbon nanotubes (p-MWCNTs). It was shown that compared with MWCNTs-COOH, p-MWCNTs induced apoptosis, reduced the level of intracellular antioxidant glutathione more significantly, and caused severer cell membrane damage as demonstrated by lactate dehydrogenase leakage. Cell cycles were arrested by both MWCNTs, while p-MWCNTs induced higher ratio of G0/G1 phase arrestment as compared with MWCNTs-COOH. Caspase-8 was also activated after both MWCNTs exposure, indicating extrinsic apoptotic pathway was involved in the apoptosis induced by MWCNTs exposure, more importantly, MWCNTs-COOH significantly reduced the activation of caspase-8 as compared with p-MWCNTs. All these results suggested that MWCNTs-COOH might be safer for in vivo application as compared with p-MWCNTs.

  15. Resveratrol Protects SAMP8 Brain Under Metabolic Stress: Focus on Mitochondrial Function and Wnt Pathway.

    PubMed

    Palomera-Avalos, V; Griñán-Ferré, C; Puigoriol-Ilamola, D; Camins, A; Sanfeliu, C; Canudas, A M; Pallàs, M

    2017-04-01

    Metabolic stress induced by high-fat (HF) diet leads to cognitive dysfunction and aging, but the physiological mechanisms are not fully understood. Senescence-accelerated prone mouse (SAMP8) models were conducted under metabolic stress conditions by feeding HF for 15 weeks, and the preventive effect of resveratrol was studied. This dietary strategy demonstrates cognitive impairment in SAMP8-HF and significant preventive effect by resveratrol-treated animals. Hippocampal changes in the proteins involved in mitochondrial dynamics optic atrophy-1 protein (OPA1) and mitofusin 2 (MFN2) comprised a differential feature found in SAMP8-HF that was prevented by resveratrol. Electronic microscopy showed a larger mitochondria in SAMP8-HF + resveratrol (SAMP8-HF + RV) than in SAMP8-HF, indicating increases in fusion processes in resveratrol-treated mice. According to the mitochondrial morphology, significant increases in the I-NDUFB8, II-SDNB, III-UQCRC2, and V-ATPase complexes, in addition to that of voltage-dependent anion channel 1 (VDAC1)/porin, were found in resveratrol-treated animals with regard to SAMP8-HF, reaching control-animal levels. Moreover, tumor necrosis factor alpha (TNF-α) and interleukin (IL-6) were increased after HF, and resveratrol prevents its increase. Moreover, we found that the HF diet affected the Wnt pathway, as demonstrated by β-catenin inactivation and modification in the expression of several components of this pathway. Resveratrol induced strong activation of β-catenin. The metabolic stress rendered in the cognitive and cellular pathways altered in SAMP8 focus on different targets in order to act on preventing cognitive impairment in neurodegeneration, and resveratrol can offer therapeutic possibilities for preventive strategies in aging or neurodegenerative conditions.

  16. Potential Dengue Virus-Triggered Apoptotic Pathway in Human Neuroblastoma Cells: Arachidonic Acid, Superoxide Anion, and NF-κB Are Sequentially Involved

    PubMed Central

    Jan, Jia-Tsrong; Chen, Bor-Horng; Ma, Shiou-Hwa; Liu, Chiu-I; Tsai, Hui-Ping; Wu, Han-Chung; Jiang, Shian-Yuan; Yang, Kuen-Der; Shaio, Men-Fang

    2000-01-01

    Direct in vivo evidence for the susceptibility of human neuronal cells to dengue virus has not been reported. In this study, we demonstrated that type 2 dengue (DEN-2) virus infection induced extensive apoptosis in the human neuroblastoma cell line SK-N-SH. Phospholipase A2 (PLA2) was activated by DEN-2 infection, which led to the generation of arachidonic acid (AA). Inhibition of PLA2 activity by the PLA2 inhibitors, AACOCF3 and ONO-RS-082, diminished DEN-2 virus-induced apoptosis. In contrast, the cyclooxygenase inhibitors aspirin and indomethacin, thought to increase AA accumulation by blocking AA catabolism, enhanced apoptosis. Exogenous AA induced apoptosis in a dose-dependent manner. Superoxide anion, which is thought to be generated through the AA-activated NADPH oxidase, was increased after infection. Pretreatment with superoxide dismutase (SOD) protected cells against DEN-2 virus-induced apoptosis. Furthermore, generation of superoxide anion was blocked by AACOCF3. In addition, the transcription factors, NF-κB and c-Jun, were found to be activated after DEN-2 virus infection. However, pretreatment of cells with oligodeoxynucleotides containing NF-κB, but not c-Jun, binding sites (transcription factor decoy) strongly prevented dengue virus-induced apoptosis. The finding that AACOCF3 and SOD significantly block activation of NF-κB suggests that this activation is derived from the AA-superoxide anion pathway. Our results indicate that DEN-2 virus infection of human neuroblastoma cells triggers an apoptotic pathway through PLA2 activation to superoxide anion generation and subsequently to NF-κB activation. This apoptotic effect can be either directly derived from the action of AA and superoxide anion on mitochondria or indirectly derived from the products of apoptosis-related genes activated by NF-κB. PMID:10954569

  17. Paeoniflorin attenuates hepatic ischemia/reperfusion injury via anti-oxidative, anti-inflammatory and anti-apoptotic pathways

    PubMed Central

    TAO, YE; WEN, ZHIHONG; SONG, YINGQIAN; WANG, HUI

    2016-01-01

    During liver surgery, hepatic blood flow needs to be blocked in order to reduce bleeding, which inevitably results in hepatic ischemia/reperfusion injury (HI/R). Paeoniflorin (PF) is the main active ingredient of the traditional Chinese herbal medicine peony, which has been shown to exert anti-oxidative and anti-apoptotic properties. In the present study, a mouse model of HI/R was generated by clamping the hepatoportal vein, hepatic artery, and hepatic duct of BALB/c mice with a vascular clamp for 30 min, followed by reperfusion for 6 h under anesthesia. Six mice in the three PF treatment groups (5, 10 and 20 mg/kg) were then injected with PF, via the tail vein. A sham group, consisting of six mice that did not undergo the procedure, and a vehicle group, consisting of 6 mice that underwent the procedure but subsequently received injections of physiological saline only, were used as controls. Liver injury was indicated by serum levels of the enzymes alanine transaminase (ALT) and aspartate transaminase (AST). The activities of oxidative stress biomarkers, including superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-PX) and malondialdehyde (MDA), were also measured. Furthermore, the activity of caspase-3 was analyzed in hepatic tissue using a commercial kit. Treatment with PF significantly attenuated HI/R injury histologically, as compared with the vehicle group. In addition, significant reductions in the serum levels of ALT and AST were observed in the PF-treated ischemic mice. Furthermore, treatment with PF enhanced the activities of hepatic tissue SOD, GSH and GSH-PX, but decreased the MDA content. Treatment of ischemic mice with PF markedly reduced the expression levels of inflammatory mediators, including nuclear factor-κB, tumor necrosis factor-α, interleukin (IL)-6, and IL-1β, and decreased the HI/R injury-induced expression of caspase-3. The results of the present study suggest that PF attenuates the HI/R injury of mice via anti

  18. RNAi-based glyconanoparticles trigger apoptotic pathways for in vitro and in vivo enhanced cancer-cell killing

    NASA Astrophysics Data System (ADS)

    Conde, João; Tian, Furong; Hernandez, Yulan; Bao, Chenchen; Baptista, Pedro V.; Cui, Daxiang; Stoeger, Tobias; de La Fuente, Jesus M.

    2015-05-01

    Gold glyconanoparticles (GlycoNPs) are full of promise in areas like biomedicine, biotechnology and materials science due to their amazing physical, chemical and biological properties. Here, siRNA GlycoNPs (AuNP@PEG@Glucose@siRNA) in comparison with PEGylated GlycoNPs (AuNP@PEG@Glucose) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo via intratracheal instillation directly into the lungs of B6 albino mice grafted with luciferase-CMT/167 adenocarcinoma cells. siRNA GlycoNPs but not PEGylated GlycoNPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose dependent manner, independent of the inflammatory response, evaluated by bronchoalveolar lavage cell counting. Moreover, in vivo pulmonary delivered siRNA GlycoNPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and apoptosis) via in vivo RNAi in tumour tissue, leading to an ~80% reduction in tumour size without associated inflammation.Gold glyconanoparticles (GlycoNPs) are full of promise in areas like biomedicine, biotechnology and materials science due to their amazing physical, chemical and biological properties. Here, siRNA GlycoNPs (AuNP@PEG@Glucose@siRNA) in comparison with PEGylated GlycoNPs (AuNP@PEG@Glucose) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo via intratracheal instillation directly into the lungs of B6 albino mice grafted with luciferase-CMT/167 adenocarcinoma cells. siRNA GlycoNPs but not PEGylated GlycoNPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose dependent manner, independent of the inflammatory response, evaluated by bronchoalveolar lavage cell counting. Moreover, in vivo pulmonary delivered siRNA GlycoNPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and

  19. Luteolin exerts pro-apoptotic effect and anti-migration effects on A549 lung adenocarcinoma cells through the activation of MEK/ERK signaling pathway.

    PubMed

    Meng, Guanmin; Chai, Kequn; Li, Xinda; Zhu, Yongqiang; Huang, Weihua

    2016-09-25

    An increasing amount of evidence suggests that luteolin, a common dietary flavonoid that is widely distributed in plants and foods, has been shown to be protective against cancer. However, the precise underlying mechanisms of its action against lung cancer are still poorly understood. In the present study, we investigated whether luteolin exhibits the anti-cancer effect in lung cancer through the induction of cell apoptosis and inhibition of cell migration, and whether mitogen-activated protein kinases (MAPKs) and Akt signaling pathways are required. Results revealed that luteolin exerted an anti-proliferation effect in a dose- and time-dependent manner in A549 lung adenocarcinoma cells, and induced apoptosis with a concomitant increase in the activation of caspases-3 and -9, diminution of Bcl-2, elevation in Bax expression, and the phosphorylation of MEK and its down-stream kinase ERK, as well as the activation of Akt. Luteolin also dramatically inhibited cell motility and migration in A549 cells. The inhibitor of MEK-ERK pathway protected against luteolin-induced cell death and suppressed the apoptosis-inducing and anti-migratory effects of luteolin, suggesting MEK-ERK signaling pathway plays an important role in mediating the pro-apoptotic effect and anti-migration effects of luteolin. Taken together, this study provides a new insight into the mode of action of luteolin on lung cancer.

  20. The Contribution of the Fas/FasL Apoptotic Pathway in Ulcer Formation during Leishmania major-Induced Cutaneous Leishmaniasis

    PubMed Central

    Eidsmo, Liv; Nylen, Susanne; Khamesipour, Ali; Hedblad, Mari-Anne; Chiodi, Francesca; Akuffo, Hannah

    2005-01-01

    Cutaneous leishmaniasis (CL), caused by the intracellular protozoan Leishmania major, is characterized by lesion formation and ulceration at the site of infection. The mechanism of ulcer formation during CL is not fully understood. The expression of Fas and FasL and the levels of apoptosis in skin biopsies and in restimulated blood mononuclear cells from patients with 1 to 7 months of L. major-induced CL were analyzed using immunohistochemistry and fluorescence-activated cell sorting analysis. The levels of soluble Fas and FasL were also analyzed by enzyme-linked immunosorbent assay. A substantial number of apoptotic keratinocytes were observed mainly in the superficial epidermis of morphologically active and healing CL skin samples. Fas expression was increased on epidermis in active CL, whereas Fas expression was similar in healing and healthy epidermis. FasL-expressing macrophages and T cells were found in subepidermal infiltrate, mainly in active disease. When CL peripheral blood mononuclear cells were restimulated with L. major, Fas was up-regulated on effector T cells, and high levels of sFasL were secreted. Supernatants from restimulated cultures induced apoptosis in human keratinocytes (HaCaT), possibly through Fas/FasL interactions. Our results indicate that FasL-expressing effector T cells and macrophages may act to induce apoptosis and ulcer formation in Fas-expressing keratinocytes during L. major infection. PMID:15793290

  1. Neuronal Ca(2+) sensor-1 contributes to stress tolerance in cardiomyocytes via activation of mitochondrial detoxification pathways.

    PubMed

    Nakamura, Tomoe Y; Nakao, Shu; Wakabayashi, Shigeo

    2016-10-01

    Identification of the molecules involved in cell death/survival pathways is important for understanding the mechanisms of cell loss in cardiac disease, and thus is clinically relevant. Ca(2+)-dependent signals are often involved in these pathways. Here, we found that neuronal Ca(2+)-sensor-1 (NCS-1), a Ca(2+)-binding protein, has an important role in cardiac survival during stress. Cardiomyocytes derived from NCS-1-deficient (Ncs1(-/-)) mice were more susceptible to oxidative and metabolic stress than wild-type (WT) myocytes. Cellular ATP levels and mitochondrial respiration rates, as well as the levels of mitochondrial marker proteins, were lower in Ncs1(-/-) myocytes. Although oxidative stress elevated mitochondrial proton leak, which exerts a protective effect by inhibiting the production of reactive oxygen species in WT myocytes, this response was considerably diminished in Ncs1(-/-) cardiomyocytes, and this would be a major reason for cell death. Consistently, H2O2-induced loss of mitochondrial membrane potential, a critical early event in cell death, was accelerated in Ncs1(-/-) myocytes. Furthermore, NCS-1 was upregulated in hearts subjected to ischemia-reperfusion, and ischemia-reperfusion injury was more severe in Ncs1(-/-) hearts. Activation of stress-induced Ca(2+)-dependent survival pathways, such as Akt and PGC-1α (which promotes mitochondrial biogenesis and function), was diminished in Ncs1(-/-) hearts. Overall, these data demonstrate that NCS-1 contributes to stress tolerance in cardiomyocytes at least in part by activating certain Ca(2+)-dependent survival pathways that promote mitochondrial biosynthesis/function and detoxification pathways.

  2. Heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell.

    PubMed

    Gu, Z T; Wang, H; Li, L; Liu, Y S; Deng, X B; Huo, S F; Yuan, F F; Liu, Z F; Tong, H S; Su, L

    2014-03-26

    Cells apoptosis induced by intense heat stress is the prominent feature of heat-related illness. However, little is known about the biological effects of heat stress on cells apoptosis. Herein, we presented evidence that intense heat stress could induce early apoptosis of HUVEC cells through activating mitochondrial pathway with changes in mitochondrial membrane potential(ΔΨm), release of cytochrome c, and activation of caspase-9 and -3. We further revealed that p53 played a crucial role in heat stress-induced early apoptosis, with p53 protein rapidly translocated into mitochondria. Using pifithrin-α(PFT), a p53's mitochondrial translocation inhibitor, we found that pretreated with PFT, heat stress induced mitochondrial p53 translocation was significantly suppressed, accompanied by a significant alleviation in the loss of ΔΨm, cytochrome c release and caspase-9 activation. Furthermore, we also found that generation of reactive oxygen species (ROS) was a critical mediator in heat stress-induced apoptosis. In addition, the antioxidant MnTMPyP significantly decreased the heat stress-induced p53's mitochondrial translocation, followed by the loss of ΔΨm, cytochrome c release, caspase-9 activation and heat stress-mediated apoptosis. Conclusively, these findings indicate the contribution of the transcription-independent mitochondrial p53 pathway to early apoptosis in HUVEC cells induced by oxidative stress in response to intense heat stress.

  3. Protective efficacy of vitamins C and E on p,p'-DDT-induced cytotoxicity via the ROS-mediated mitochondrial pathway and NF-κB/FasL pathway.

    PubMed

    Jin, Xiaoting; Song, Li; Liu, Xiangyuan; Chen, Meilan; Li, Zhuoyu; Cheng, Long; Ren, Hua

    2014-01-01

    Dichlorodiphenoxytrichloroethane (DDT) is a known persistent organic pollutant and liver damage toxicant. However, there has been little emphasis on the mechanism underlying liver damage toxicity of DDT and the relevant effective inhibitors. Hence, the present study was conducted to explore the protective effects of vitamin C (VC) and vitamin E (VE) on the cytotoxicity of DDT in HL-7702 cells and elaborate the specific molecular mechanisms. The results demonstrated that p,p'-DDT exposure at over 10 µM depleted cell viability of HL-7702 cells and led to cell apoptotic. p,p'-DDT treatment elevated the level of reactive oxygen species (ROS) generation, induced mitochondrial membrane potential, and released cytochrome c into the cytosol, with subsequent elevations of Bax and p53, along with suppression of Bcl-2. In addition, the activations of caspase-3 and -8 were triggered. Furthermore, p,p'-DDT promoted the expressions of NF-κB and FasL. When the cells were exposed to the NF-κB inhibitor (PDTC), the up-regulated expression of FasL was attenuated. Strikingly, these alterations caused by DDT treatment were prevented or reversed by the addition of VC or VE, and the protective effects of co-treatment with VC and VE were higher than the single supplement with p,p'-DDT. Taken together, these findings provide novel experimental evidences supporting that VC or/and VE could reduce p,p'-DDT-induced cytotoxicity of HL-7702 cells via the ROS-mediated mitochondrial pathway and NF-κB/FasL pathway.

  4. Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis.

    PubMed

    Wilkins, Heather M; Harris, Janna L; Carl, Steven M; E, Lezi; Lu, Jianghua; Eva Selfridge, J; Roy, Nairita; Hutfles, Lewis; Koppel, Scott; Morris, Jill; Burns, Jeffrey M; Michaelis, Mary L; Michaelis, Elias K; Brooks, William M; Swerdlow, Russell H

    2014-12-15

    Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis.

  5. Natural pyrethrins induces apoptosis in human hepatocyte cells via Bax- and Bcl-2-mediated mitochondrial pathway.

    PubMed

    Yang, Yun; Zong, Mimi; Xu, Wenping; Zhang, Yang; Wang, Bo; Yang, Mingjun; Tao, Liming

    2017-01-25

    Natural pyrethrins have been widely used for pest control in organic farming and for residential indoor pest managements. Although the specific mechanisms underlying their activity are incompletely understood, natural pesticides are considered the safest based on their target specificity and rapid degradation in the environment. Here, we used in vitro bioassays to characterize the cytotoxic effects of natural pyrethrins and attempted to delineate the cellular and molecular mechanisms of their cytotoxicity against human hepatocytes. The results demonstrate that natural pyrethrins reduce cell viability and enhance apoptosis in HepG2 cells. In addition, the current data indicate that natural pyrethrins cause a reduction in the mitochondrial membrane potential (Δψm), increase reactive oxygen species production, and up-regulate the Bax/Bcl-2 expression, leading to the release of cytochrome-c into the cytosol, activation of caspase-9 and caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). Taken together, the results indicate that natural pyrethrins has potentially exert adverse effects on human health by inducing caspase-dependent apoptosis in hepatocytes through Bax- and Bcl-2-mediated mitochondrial pathway.

  6. Fipronil induces apoptosis through caspase-dependent mitochondrial pathways in Drosophila S2 cells.

    PubMed

    Zhang, Baoyan; Xu, Zhiping; Zhang, Yixi; Shao, Xusheng; Xu, Xiaoyong; Cheng, Jiaogao; Li, Zhong

    2015-03-01

    Fipronil is the first phenylpyrazole insecticide widely used in controlling pests, including pyrethroid, organophosphate and carbamate insecticides. It is generally accepted that fipronil elicits neurotoxicity via interactions with GABA and glutamate receptors, although alternative mechanisms have recently been proposed. This study evaluates the genotoxicity of fipronil and its likely mode of action in Drosophila S2 cells, as an in vitro model. Fipronil administrated the concentration- and time-dependent S2 cell proliferation. Intracellular biochemical assays showed that fipronil-induced S2 cell apoptosis coincided with a decrease in the mitochondrial membrane potential and an increase reactive oxygen species generation, a significant decrease of Bcl-2 and DIAP1, and a marked augmentation of Cyt c and caspase-3. Because caspase-3 is the major executioner caspase downstream of caspase-9 in Drosophila, enzyme activity assays were used to determine the activities of caspase-3 and caspase-9. Our results indicated that fipronil effectively induced apoptosis in Drosophila S2 cells through caspase-dependent mitochondrial pathways.

  7. DJ-1 ameliorates ischemic cell death in vitro possibly via mitochondrial pathway.

    PubMed

    Kaneko, Yuji; Shojo, Hideki; Burns, Jack; Staples, Meaghan; Tajiri, Naoki; Borlongan, Cesar V

    2014-02-01

    DJ-1 is an important redox-reactive neuroprotective protein implicated in regulation of oxidative stress after ischemia. However the molecular mechanism, especially the mitochondrial function, by which DJ-1 protects neuronal cells in stroke remains to be elucidated. The aim of this study was to reveal whether DJ-1 translocates into the mitochondria in exerting neuroprotection against an in vitro model of stroke. Human neural progenitor cells (hNPCs) were initially exposed to oxygen-glucose deprivation and reperfusion injury, and thereafter, DJ-1 translocation was measured by immunocytochemistry and its secretion by hNPCs was detected by enzyme-linked immunosorbant assay (ELISA). Exposure of hNPCs to experimental stroke injury resulted in DJ-1 translocation into the mitochondria. Moreover, significant levels of DJ-1 protein were secreted by the injured hNPCs. Our findings revealed that DJ-1 principally participates in the early phase of stroke involving the mitochondrial pathway. DJ-1 was detected immediately after stroke and efficiently translocated into the mitochondria offering a new venue for developing treatment strategies against ischemic stroke.

  8. Thymineless Death in F10-Treated AML Cells Occurs via Lipid Raft Depletion and Fas/FasL co-Localization in the Plasma Membrane with Activation of the Extrinsic Apoptotic Pathway

    PubMed Central

    Gmeiner, William H.; Jennings-Gee, Jamie; Stuart, Christopher H.; Pardee, Timothy S.

    2014-01-01

    The polymeric fluoropyrimidine F10 displays excellent anti-leukemia activity in pre-clinical models of acute myelogenous leukemia (AML) through dual targeting of thymidylate synthase and DNA topoisomerase 1. Here we report that F10 activates the extrinsic apoptotic pathway in AML cells by enhancing localization of Fas and Fas ligand (FasL) at the plasma membrane and while reducing overall lipid raft levels promotes Fas/FasL co-localization in remaining lipid rafts. The HMG-CoA synthase inhibitor simvastatin was synergistic with F10 and induced cell death via similar apoptotic processes. Our results are consistent with diverse processes activating a common apoptotic pathway characterized by reduced overall levels of lipid rafts and Fas/FasL co-localization in the plasma membrane, including in remaining lipid rafts which may play a role in both cell-survival and cell death signaling. PMID:25510486

  9. Thymineless death in F10-treated AML cells occurs via lipid raft depletion and Fas/FasL co-localization in the plasma membrane with activation of the extrinsic apoptotic pathway.

    PubMed

    Gmeiner, William H; Jennings-Gee, Jamie; Stuart, Christopher H; Pardee, Timothy S

    2015-02-01

    The polymeric fluoropyrimidine F10 displays excellent anti-leukemia activity in pre-clinical models of acute myelogenous leukemia (AML) through dual targeting of thymidylate synthase and DNA topoisomerase 1. Here we report that F10 activates the extrinsic apoptotic pathway in AML cells by enhancing localization of Fas and Fas ligand (FasL) at the plasma membrane and while reducing overall lipid raft levels promotes Fas/FasL co-localization in remaining lipid rafts. The HMG-CoA synthase inhibitor simvastatin was synergistic with F10 and induced cell death via similar apoptotic processes. Our results are consistent with diverse processes activating a common apoptotic pathway characterized by reduced overall levels of lipid rafts and Fas/FasL co-localization in the plasma membrane, including in remaining lipid rafts which may play a role in both cell-survival and cell death signaling.

  10. Stimulation of apoptotic pathways in liver cancer cells: An alternative perspective on the biocompatibility and the utility of biomedical glasses.

    PubMed

    Kilcup, Nancy; Gaynard, Seán; Werner-Zwanziger, Ulrike; Tonkopi, Elena; Hayes, Jessica; Boyd, Daniel

    2016-05-01

    A host of research opportunities with innumerable clinical applications are open to biomedical glasses if one considers their potential as therapeutic inorganic ion delivery systems. Generally, applications have been limited to repair and regeneration of hard tissues while compositions are largely constrained to the original bioactive glass developed in the 1960s. However, in oncology applications the therapeutic paradigm shifts from repair to targeted destruction. With this in mind, the composition-structure-property-function relationships of vanadium-containing zinc-silicate glasses (0.51SiO2-0.29Na2O-(0.20-X)ZnO-XV2O5, 0 ≤ X ≤ 0.09) were characterized in order to determine their potential as therapeutic inorganic ion delivery systems. Increased V2O5mole fraction resulted in a linear decrease in density and glass transition temperature (Tg).(29)Si MAS NMR peak maxima shifted upfield while(51)V MAS NMR peak maxima were independent of V2O5content and overlapped well with the spectra NaVO3 Increased V2O5mole fraction caused ion release to increase. When human liver cancer cells, HepG2, were exposed to these ions they demonstrated a concentration-dependent cytotoxic response, mediated by apoptosis. This work demonstrates that the zinc-silicate system studied herein is capable of delivering therapeutic inorganic ions at concentrations that induce apoptotic cell death and provide a simple means to control therapeutic inorganic ion delivery.

  11. Antiapoptotic and Antioxidant Properties of Orthosiphon stamineus Benth (Cat's Whiskers): Intervention in the Bcl-2-Mediated Apoptotic Pathway

    PubMed Central

    Abdelwahab, Siddig Ibrahim; Mohan, Syam; Mohamed Elhassan, Manal; Al-Mekhlafi, Nabil; Mariod, Abdelbasit Adam; Abdul, Ahmad Bustamam; Abdulla, Mahmood Ameen; Alkharfy, Khalid M.

    2011-01-01

    Antiapoptotic and antioxidant activities of aqueous-methanolic extract (CAME) of Orthosiphonstamineus Benth(OS), and its hexane (HF), chloroform (CF), n-butanol (NBF), ethyl acetate (EAF) and water (WF) fractions were investigated. Antioxidant properties were evaluated using the assays of Folin-Ciocalteu, aluminiumtrichloride, β-carotene bleaching and DPPH. The role of OS against hydrogen peroxide induced apoptosis on MDA-M231 epithelial cells was examined using MTT assay, phase contrast microscope, colorimetric assay of caspase-3, western blot and quantitative real-time PCR. Results showed that EAF showed the highest total phenolic content followed by CAME, NBF, WF, CF and HF, respectively. Flavonoid content was in the order of the CF > EAF > HF > CAME > NBF > WF. The IC50 values on DPPH assay for different extract/fractions were 126.2 ± 23, 31.25 ± 1.2, 15.25 ± 2.3, 13.56 ± 1.9, 23.0 ± 3.2, and 16.66 ± 1.5 μg/ml for HF, CF, EAF, NBF, WF and CAME, respectively. OSreduced the oxidation of β-carotene by hydroperoxides. Cell death was dose-dependently inhibited by pretreatment with OS. Caspase-3 and distinct morphological features suggest the anti-apoptotic activities of OS. This plant not only increased the expression of Bcl-2, but also decreased Bax expression, and ultimately reduced H2O2-induced apoptosis. The current results showed that phenolics may provide health and nutritional benefits. PMID:21234328

  12. Luffa echinata Roxb. Induced Apoptosis in Human Colon Cancer Cell (SW-480) in the Caspase-dependent Manner and Through a Mitochondrial Apoptosis Pathway

    PubMed Central

    Shang, Li-Hua; Yu, Yan; Che, De-Hai; Pan, Bo; Jin, Shi; Zou, Xiao-Long

    2016-01-01

    Background: Luffa echinata Roxb. (LER) (Cucurbitaceae) showed tremendous medicinal importance and are being used for the treatment of different ailments. Objective: In this study, the antiproliferative properties and cell death mechanism induced by the extract of the fruits of LER were investigated. Materials and Methods: MTT and LDH assay were used to test the antiproliferative and cytotoxicity of LER extract, respectively. The intracellular ROS were measured by a fluorometric assay. The expression of several apoptotic-related proteins in SW-480 cells treated by LER was evaluated by Western blot analysis. Results: The methanolic extract of LER fruits inhibited the proliferation of human colon cancer cells (SW-480) in both dose- and time-dependent manners. The LER-treated cells showed obvious characteristics of cell apoptosis, including cell shrinkage, destruction of the monolayer, and condensed chromatin. In addition, treatments of various concentrations of LER extracts caused the release of lactate dehydrogenase as a dose-dependent manner via stimulation of the intracellular metabolic system. LER induced apoptosis, DNA fragmentation, and cellular ROS accumulation in SW-480 cells. Treatment of LER on SW-480 cells promoted the expression of caspases, Bax, Bad, and p53 proteins and decreased the levels of Bcl-2 and Bcl-XL. Conclusions: These results indicated that treatment with LER-induced cell death in mitochondrial apoptosis pathway by regulating pro-apoptotic proteins via the up regulation of the p53 protein. These findings highlight the potentials of LER in the treatment of human colon cancer. SUMMARY LER induced apoptosis, DNA fragmentation, and cellular ROS accumulation in SW-480 cells. Treatment of LER on SW-480 cells promoted the expression of caspases, Bax, Bad, and p53 proteins and decreased the levels of Bcl-2 and Bcl-XL. PMID:27019558

  13. Mechanisms of cell death pathway activation following drug-induced inhibition of mitochondrial complex I

    PubMed Central

    Imaizumi, Naoki; Kwang Lee, Kang; Zhang, Carmen; Boelsterli, Urs A.

    2015-01-01

    Respiratory complex I inhibition by drugs and other chemicals has been implicated as a frequent mode of mitochondria-mediated cell injury. However, the exact mechanisms leading to the activation of cell death pathways are incompletely understood. This study was designed to explore the relative contributions to cell injury of three distinct consequences of complex I inhibition, i.e., impairment of ATP biosynthesis, increased formation of superoxide and, hence, peroxynitrite, and inhibition of the mitochondrial protein deacetylase, Sirt3, due to imbalance of the NADH/NAD+ ratio. We used the antiviral drug efavirenz (EFV) to model drug-induced complex I inhibition. Exposure of cultured mouse hepatocytes to EFV resulted in a rapid onset of cell injury, featuring a no-effect level at 30 µM EFV and submaximal effects at 50 µM EFV. EFV caused a concentration-dependent decrease in cellular ATP levels. Furthermore, EFV resulted in increased formation of peroxynitrite and oxidation of mitochondrial protein thiols, including cyclophilin D (CypD). This was prevented by the superoxide scavenger, Fe-TCP, or the peroxynitrite decomposition catalyst, Fe-TMPyP. Both ferroporphyrins completely protected from EFV-induced cell injury, suggesting that peroxynitrite contributed to the cell injury. Finally, EFV increased the NADH/NAD+ ratio, inhibited Sirt3 activity, and led to hyperacetylated lysine residues, including those in CypD. However, hepatocytes isolated from Sirt3-null mice were protected against 40 µM EFV as compared to their wild-type controls. In conclusion, these data are compatible with the concept that chemical inhibition of complex I activates multiple pathways leading to cell injury; among these, peroxynitrite formation may be the most critical. PMID:25625582

  14. Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

    SciTech Connect

    Sheng Zhiguo; Cao Xiaojuan; Peng Shuangqing Wang Changyong; Li Qianqian; Wang Yimei; Liu Mifeng

    2008-01-15

    Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the {beta}{sub 1} integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes.

  15. 7-O-Geranylquercetin induces apoptosis in gastric cancer cells via ROS-MAPK mediated mitochondrial signaling pathway activation.

    PubMed

    Zhu, Yanyan; Jiang, Yameng; Shi, Lei; Du, Linying; Xu, Xiaodong; Wang, Enxia; Sun, Yong; Guo, Xin; Zou, Boyang; Wang, Huaxin; Wang, Changyuan; Sun, Lidan; Zhen, Yuhong

    2017-03-01

    7-O-Geranylquercetin (GQ) is a novel O-alkylated derivate of quercetin. In this study, we evaluated its apoptosis induction effects in human gastric cancer cell lines SGC-7901 and MGC-803 and explored the potential molecular mechanisms. The results demonstrated that GQ lowered viability of SGC-7901 and MGC-803 cells in a dose- and time-dependent manner without apparent cytotoxicity to human gastric epithelial cell line GES-1. GQ could induce apoptosis in SGC-7901 and MGC-803cells, and arrest the gastric cancer cells at G2/M phase. Mechanism study showed that GQ triggered generation of reactive oxygen species (ROS), then activated p38 and JNK signaling pathways, subsequently led to mitochondrial impairment by regulating the expression of Bcl-2, Bcl-xl and Bax, and finally promoted the release of cytochrome c and the activation of caspases to induce apoptosis. In addition, Z-VAD-FMK (caspase inhibitor) could reverse GQ-induced apoptosis. SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) could rescue GQ-induced cell death and attenuate mitochondrial signal pathway activation. Furthermore, NAC (ROS inhibitor) could rescue GQ-induced cell death, reduce ROS generation, decrease the phosphorylation of p38 and JNK, and then attenuate the activation of mitochondrial signal pathway. Taken together, GQ induces caspase-dependent apoptosis in gastric cancer cells through activating ROS-MAPK mediated mitochondrial signal pathway. This study highlights the potential use of GQ as a gastric cancer therapeutic agent.

  16. Activation of a Novel Death Pathway, Targeted Necrosis, by p53 Peptides to Circumvent Apoptotic Resistance in Prostate Cancer

    DTIC Science & Technology

    2010-10-01

    Virtually all chemotherapy agents, at clinically achievable concentrations, act by inducing cancer cell death via apoptosis, but cancer cells...prostate cancer cells by induction of ?targeted necrosis?. Targeted necrosis has potential clinical utility, since its cell death mechanism retains...Therefore, pretreatment with paclitaxel may prime the cells by induction of Fas ligand so that subsequent exposure to p53p-Ant efficiently activates the Fas/FADD cell death pathway.

  17. Resveratrol Sensitizes Acute Myelogenous Leukemia Cells to Histone Deacetylase Inhibitors through Reactive Oxygen Species-Mediated Activation of the Extrinsic Apoptotic PathwayS⃞

    PubMed Central

    Yaseen, Alae; Chen, Shuang; Hock, Stefanie; Rosato, Roberto; Dent, Paul; Dai, Yun

    2012-01-01

    Histone deacetylase inhibitors (HDACIs) activate the prosurvival nuclear factor-κB (NF-κB) pathway by hyperacetylating RelA/p65, whereas the chemopreventive agent resveratrol inhibits NF-κB by activating the class III histone deacetylase Sirt1. Interactions between resveratrol and pan-HDACIs (vorinostat and panobinostat) were examined in human acute myelogenous leukemia (AML) cells. Pharmacologically achievable resveratrol concentrations (25–50 μM) synergistically potentiated HDACI lethality in AML cell lines and primary AML blasts. Resveratrol antagonized RelA acetylation and NF-κB activation in HDACI-treated cells. However, short hairpin RNA Sirt1 knockdown failed to modify HDACI sensitivity, which suggests that factors other than or in addition to Sirt1 activation contribute to resveratrol/HDACI interactions. These interactions were associated with death receptor 5 (DR5) up-regulation and caspase-8 activation, whereas cells expressing dominant-negative caspase-8 were substantially protected from resveratrol/HDACI treatment, which suggests a significant functional role for the extrinsic apoptotic pathway in lethality. Exposure to resveratrol with HDACI induced sustained reactive oxygen species (ROS) generation, which was accompanied by increased levels of DNA double-strand breaks, as reflected in γH2A.X and comet assays. The free radical scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin chloride blocked ROS generation, DR5 up-regulation, caspase-8 activation, DNA damage, and apoptosis, which indicates a primary role for oxidative injury in lethality. Analyses of cell-cycle progression and 5-ethynyl-2′-deoxyuridine incorporation through flow cytometry revealed that resveratrol induced S-phase accumulation; this effect was abrogated by HDACI coadministration, which suggests that cells undergoing DNA synthesis may be particularly vulnerable to HDACI lethality. Collectively, these findings indicate that resveratrol interacts synergistically with HDACIs in

  18. Molecular mechanisms of (-)-epicatechin and chlorogenic acid on the regulation of the apoptotic and survival/proliferation pathways in a human hepatoma cell line.

    PubMed

    Granado-Serrano, Ana Belén; Martín, María Angeles; Izquierdo-Pulido, María; Goya, Luis; Bravo, Laura; Ramos, Sonia

    2007-03-07

    Dietary polyphenols have been associated with reduced risk of chronic diseases, but the precise molecular mechanisms of protection remain unclear. This work was aimed at studying the effect of (-)-epicatechin (EC) and chlorogenic acid (CGA) on the regulation of apoptotic and survival/proliferation pathways in a human hepatoma cell line (HepG2). EC or CGA treatment for 18 h had a slight effect on cell viability and decreased reactive oxygen species formation, and EC alone promoted cell proliferation, whereas CGA increased glutathione levels. Phenols neither induced the caspase cascade for apoptosis nor affected expression levels of Bcl-xL or Bax. A sustained activation of the major survival signals AKT/PI-3-kinase and ERK was shown in EC-treated cells, rather than in CGA-exposed cells. These data suggest that EC and CGA have no effect on apoptosis and enhance the intrinsic cellular tolerance against oxidative insults either by activating survival/proliferation pathways or by increasing antioxidant potential in HepG2.

  19. Anticancer drugs induce hypomethylation of the acetylcholinesterase promoter via a phosphorylated-p38-DNMT1-AChE pathway in apoptotic hepatocellular carcinoma cells.

    PubMed

    Xi, Qiliang; Gao, Ning; Yang, Yang; Ye, Weiyuan; Zhang, Bo; Wu, Jun; Jiang, Gening; Zhang, Xuejun

    2015-11-01

    Apoptosis, also known as programmed cell death, plays an essential role in eliminating excessive, damaged or harmful cells. Previous work has demonstrated that anticancer drugs induce cell apoptosis by inducing cytotoxicity. In recent years, several reports demonstrated modulated expression of DNA methyltransferases 1 (DNMT1) and acetylcholinesterase (AChE) in a variety of tumors. In this study, we showed that the expression of DNMT1 was decreased and the methylation of CpGs in the promoter of AChE was reduced in anticancer drugs-induced apoptotic hepatocellular carcinoma cells. Silencing of DNMT1 expression by AZA or RNA interference (RNAi) restored AChE production and inhibition of AChE expression by RNAi protected HCC cells from anticancer drugs-induced apoptosis. Furthermore, we demonstrated that the regulation of AChE by DNMT1 was involved in the phosphorylated p38 pathway in anticancer drugs-induced apoptosis. In addition, immunohistochemical staining showed that P-p38, DNMT1 and AChE were aberrantly expressed in a subset of HCC tumors. Taken together, we demonstrated the regulation of AChE by DNMT1 and further, we found that this regulation was involved in the phosphorylated p38 pathway in anticancer drugs-induced apoptosis.

  20. Cytotoxicity of 11-epi-Sinulariolide Acetate Isolated from Cultured Soft Corals on HA22T Cells through the Endoplasmic Reticulum Stress Pathway and Mitochondrial Dysfunction

    PubMed Central

    Lin, Jen-Jie; Wang, Robert Y. L.; Chen, Jiing-Chuan; Chiu, Chien-Chih; Liao, Ming-Hui; Wu, Yu-Jen

    2016-01-01

    Natural compounds from soft corals have been increasingly used for their antitumor therapeutic properties. This study examined 11-epi-sinulariolide acetate (11-epi-SA), an active compound isolated from the cultured soft coral Sinularia flexibilis, to determine its potential antitumor effect on four hepatocellular carcinoma cell lines. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the results demonstrated that 11-epi-SA treatment showed more cytotoxic effect toward HA22T cells. Protein profiling of the 11-epi-SA-treated HA22T cells revealed substantial protein alterations associated with stress response and protein synthesis and folding, suggesting that the mitochondria and endoplasmic reticulum (ER) play roles in 11-epi-SA-initiated apoptosis. Moreover, 11-epi-SA activated caspase-dependent apoptotic cell death, suggesting that mitochondria-related apoptosis genes were involved in programmed cell death. The unfolded protein response signaling pathway-related proteins were also activated on 11-epi-SA treatment, and these changes were accompanied by the upregulated expression of growth arrest and DNA damage-inducible protein (GADD153) and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), the genes encoding transcription factors associated with growth arrest and apoptosis under prolonged ER stress. Two inhibitors, namely salubrinal (Sal) and SP600125, partially abrogated 11-epi-SA-related cell death, implying that the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)–activating transcription factor (ATF) 6–CHOP or the inositol-requiring enzyme 1 alpha (IRE1α)–c-Jun N-terminal kinase (JNK)–cJun signal pathway was activated after 11-epi-SA treatment. In general, these results suggest that 11-epi-SA exerts cytotoxic effects on HA22T cells through mitochondrial dysfunction and ER stress cell death pathways. PMID:27801783

  1. Induction of G2/M phase cell cycle arrest and apoptosis by ginsenoside Rf in human osteosarcoma MG‑63 cells through the mitochondrial pathway.

    PubMed

    Shangguan, Wen-Ji; Li, He; Zhang, Yue-Hui

    2014-01-01

    Ginsenosides, extracted from the traditional Chinese herb ginseng, are a series of novel natural anticancer products known for their favorable safety and efficacy profiles. The present study aimed to investigate the cytotoxicity of ginsenoside Rf to human osteosarcoma cells and to explore the anticancer molecular mechanisms of ginsenoside Rf. Five human osteosarcoma cell lines (MG-63, OS732, U-2OS, HOS and SAOS-2) were employed to investigate the cytotoxicity of ginsenoside Rf by MTT and colony forming assays. After treatment with ginsenoside Rf, MG-63 cells which were the most sensitive to ginsenoside Rf, were subjected to flow cytometry to detect cell cycle distribution and apoptosis, and nuclear morphological changes were visualized by Hoechst 33258 staining. Caspase-3, -8 and -9 activities were also evaluated. The expression of cell cycle markers including cyclin B1 and Cdk1 was detected by RT-PCR and western blotting. The expression of apoptotic genes Bcl-2 and Bax and the release of cytochrome c were also examined by western blotting. Change in the mitochondrial membrane potential was observed by JC-1 staining in situ. Our results demonstrated that the cytotoxicity of ginsenoside Rf to these human osteosarcoma cell lines was dose-dependent, and the MG-63 cells were the most sensitive to exposure to ginsenoside Rf. Additionally, ginsenoside Rf induced G2/M phase cell cycle arrest and apoptosis in MG-63 cells. Furthermore, we observed upregulation of Bax and downregulation of Bcl-2, Cdk1 and cyclin B1, the activation of caspase-3 and -9 and the release of cytochrome c in MG-63 cells following treatment with ginsenoside Rf. Our findings demonstrated that ginsenoside Rf induces G2/M phase cell cycle arrest and apoptosis in human osteosarcoma MG-63 cells through the mitochondrial pathway, suggesting that ginsenoside Rf, as an effective natural product, may have a therapeutic effect on human osteosarcoma.

  2. Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells.

    PubMed

    Arbon, Kate S; Christensen, Cody M; Harvey, Wendy A; Heggland, Sara J

    2012-02-01

    Recent reports of cadmium in electronic waste and jewelry have increased public awareness regarding this toxic metal. Human exposure to cadmium is associated with the development of osteoporosis. We previously reported cadmium induces apoptosis in human tumor-derived Saos-2 osteoblasts. In this study, we examine the extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC) pathways in cadmium-induced apoptosis and altered osteoblast gene expression. Saos-2 osteoblasts were cultured in the presence or absence of 10μM CdCl(2) for 2-72h. We detected significant ERK activation in response to CdCl(2) and pretreatment with the ERK inhibitor PD98059 attenuated cadmium-induced apoptosis. However, PKCα activation was not observed after exposure to CdCl(2) and pretreatment with the PKC inhibitor, Calphostin C, was unable to rescue cells from cadmium-induced apoptosis. Gene expression studies were conducted using qPCR. Cells exposed to CdCl(2) exhibited a significant decrease in the bone-forming genes osteopontin (OPN) and alkaline phosphatase (ALP) mRNA. In contrast, SOST, whose protein product inhibits bone formation, significantly increased in response to CdCl(2). Pretreatment with PD98059 had a recovery effect on cadmium-induced changes in gene expression. This research demonstrates cadmium can directly inhibit osteoblasts via ERK signaling pathway and identifies SOST as a target for cadmium-induced osteotoxicity.

  3. Disulfiram induces copper-dependent stimulation of reactive oxygen species and activation of the extrinsic apoptotic pathway in melanoma.

    PubMed

    Morrison, Brian W; Doudican, Nicole A; Patel, Kirtesh R; Orlow, Seth J

    2010-02-01

    Melanoma is the most aggressive and deadly form of skin cancer. The current standard of care produces response rates of less than 20%, underscoring the critical need for identification of new effective, nontoxic therapies. Disulfiram (DSF) was identified using a drug screen as one of the several compounds that preferentially decreased proliferation in multiple melanoma subtypes compared with benign melanocytes. DSF, a member of the dithiocarbamate family, is a copper (Cu) chelator, and Cu has been shown previously to enhance DSF-mediated growth inhibition and apoptosis in cancer cells. Here, we report that in the presence of free Cu, DSF inhibits cellular proliferation and induces apoptosis in a panel of cell lines representing primary and metastatic nodular and superficial spreading melanoma. Both decreased cellular proliferation and increased apoptosis were seen at 50-500 nmol/l DSF concentrations that are achievable through oral dosing of the medication. In the presence of Cu, DSF caused activation of the extrinsic pathway of apoptosis as measured by caspase-8 cleavage. The addition of Z-IETD-FMK, a selective caspase-8 inhibitor, was protective against DSF-Cu-induced apoptosis. Production of reactive oxygen species (ROS) in response to DSF-Cu treatment preceded the induction of apoptosis. Both ROS production and apoptosis were prevented by coincubation of N-acetyl cysteine, a free radical scavenger. Our study shows that DSF might be used to target both nodular and superficial spreading melanoma through ROS production and activation of the extrinsic pathway of apoptosis.

  4. A novel benzothiazole derivative YLT322 induces apoptosis via the mitochondrial apoptosis pathway in vitro with anti-tumor activity in solid malignancies.

    PubMed

    Xuejiao, Song; Yong, Xia; Ningyu, Wang; Lidan, Zhang; Xuanhong, Shi; Youzhi, Xu; Tinghong, Ye; Yaojie, Shi; Yongxia, Zhu; Luoting, Yu

    2013-01-01

    Benzothiazole derivatives are known for various biological activities, and their potency in cancer therapy has received considerable attention in recent years. YLT322, a novel synthesized benzothiazole derivative, exhibits potent anti-tumor activity via inducing apoptosis both in vitro and in vivo. In this study, we found that YLT322 showed growth inhibition against a broad spectrum of human cancer cells and induced apoptosis of HepG2 cells in a dose- and time-dependent manner. The occurrence of its apoptosis was associated with activation of caspases-3 and -9, but not caspase-8. YLT322 increased the expression of Bax, decreased the expression of Bcl-2, and induced the release of cytochrome c which activates the mitochondrial apoptotic pathway. The down-regulation of phosphorylated p42/44 MAPK and phosphorylated Akt was also observed. Moreover, YLT322 suppressed the growth of established tumors in xenograft models in mice without obvious side effects. Histological and immunohistochemical analyses revealed an increase in TUNEL and caspase-3-positive cells and a decrease in Ki67-positive cells upon YLT322. These results suggest that YLT322 may be a potential candidate for cancer therapy.

  5. Cherry Valley Ducks Mitochondrial Antiviral-Signaling Protein-Mediated Signaling Pathway and Antiviral Activity Research

    PubMed Central

    Li, Ning; Hong, Tianqi; Li, Rong; Wang, Yao; Guo, Mengjiao; Cao, Zongxi; Cai, Yumei; Liu, Sidang; Chai, Tongjie; Wei, Liangmeng

    2016-01-01

    Mitochondrial antiviral-signaling protein (MAVS), an adaptor protein of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)-mediated signal pathway, is involved in innate immunity. In this study, Cherry Valley duck MAVS (duMAVS) was cloned from the spleen and analyzed. duMAVS was determined to have a caspase activation and recruitment domain at N-terminal, followed by a proline-rich domain and a transmembrane domain at C-terminal. Quantitative real-time PCR indicated that duMAVS was expressed in all tissues tested across a broad expression spectrum. The expression of duMAVS was significantly upregulated after infection with duck Tembusu virus (DTMUV). Overexpression of duMAVS could drive the activation of interferon (IFN)-β, nuclear factor-κB, interferon regulatory factor 7, and many downstream factors (such as Mx, PKR, OAS, and IL-8) in duck embryo fibroblast cells. What is more, RNA interference further confirmed that duMAVS was an important adaptor for IFN-β activation. The antiviral assay showed that duMAVS could suppress the various viral replications (DTMUV, novel reovirus, and duck plague virus) at early stages of infection. Overall, these results showed that the main signal pathway mediated by duMAVS and it had a broad-spectrum antiviral ability. This research will be helpful to better understanding the innate immune system of ducks. PMID:27708647

  6. High CerS5 expression levels associate with reduced patient survival and transition from apoptotic to autophagy signalling pathways in colorectal cancer

    PubMed Central

    Fitzgerald, Seán; Sheehan, Katherine M; Espina, Virginia; O'Grady, Anthony; Cummins, Robert; Kenny, Dermot; Liotta, Lance; O'Kennedy, Richard; Kay, Elaine W

    2014-01-01

    Abstract Ceramide synthase 5 is involved in the de novo synthesis of ceramide, a sphingolipid involved in cell death and proliferation. In this study, we investigated the role of ceramide synthase 5 in colorectal cancer by examining ceramide synthase 5 expression, clinico‐pathological parameters and association with survival/death signalling pathways in cancer. Immunohistochemical analysis of CerS5 was performed on 102 colorectal cancer samples using tissue microarrays constructed from formalin‐fixed and paraffin‐embedded tissues. We found strong membranous ceramide synthase 5 staining in 57 of 102 (56%) colorectal cancers. A multivariate Cox regression analysis of ceramide synthase 5 expression adjusted for disease stage, differentiation and lymphovascular invasion revealed reduced 5‐year overall survival (p = 0.001) and 5‐year recurrence‐free survival (p = 0.002), with hazard ratios of 4.712 and 4.322, respectively. The effect of ceramide synthase 5 expression on tumourigenic processes was further characterised by reverse phase protein array analysis. Reverse phase protein arrays were generated from laser capture microdissection‐enriched carcinoma cells from 19 fresh‐frozen colorectal cancer tissues. Measurements of phosphorylation and total levels of signalling proteins involved in apoptosis, autophagy and other cancer‐related pathways revealed two distinct signalling networks; weak membranous ceramide synthase 5 intensity was associated with a proteomic network dominated by signalling proteins linked to apoptosis, whereas strong ceramide synthase 5 intensity was associated with a proteomic sub‐network mostly composed of proteins linked to autophagy. In conclusion, high ceramide synthase 5 expression was found in colorectal cancer tissue and was associated with poorer patient outcomes. Our findings suggest that this may be mediated by a transition from apoptotic to autophagy signalling pathways in ceramide synthase 5 High expressing

  7. EXTRINSIC APOPTOTIC PATHWAYS: A NEW POTENTIAL “TARGET” FOR MORE SUFFICIENT THERAPY IN A CASE OF CUTANEOUS ANAPLASTIC LARGE CD30+ ALK–T-CELL LYMPHOMA

    PubMed Central

    Georgi, Tchernev; Carlos, Cardoso José; Lubomir, Arseniev; Hiroyuki, Okamoto

    2011-01-01

    The primary cutaneous T-cell lymphomas (CTCL) represent a clonal T-lymphocyte proliferation infiltrating the skin. CD30+T-cell lymphomas present clinically as nodules with a diameter between 1 and 15 cm, mostly in elderly patients. The role of the CD30 molecule in patients suffering from T-cell lymphomas is not completely clear yet. The signal transduction pathway which includes CD30 seems to play a key role in tumor progression. In certain forms of T-cellular lymphomas, the interaction between CD30/CD30-ligand is able to provoke apoptosis of the “tumor lymphocytes”. The modern conceptions of the pathogenesis of T-cell lymphomas include disorders in the pathways involved in programmed cellular death and disregulation in the expression of certain of its regulatory molecules. We are presenting an unusual case of a female patient with a primary cutaneous form of CD30+/ALK− anaplastic large T-cell lymphoma. Upon the introduction of systemic PUVA, (psoralen plus ultraviolet light radiation) combined with beam therapy, a complete remission could be noticed. Eight months later, we observed a local recurrence, which was overcome by CHOP chemotherapy (Cyclophosphamide, Hydroxydaunorubicin (Doxorubicin), Vincristin (Oncovin®), Predniso(lo)n). Six months later, new cutaneous lesions had been noticed again. A new therapeutic hope for the patients with anaplastic large CTCL is actually based on the influence of the activity of the different apoptotic pathways. Death ligands, including tumor necrosis factor (TNF)-α, CD95L/FasL, and TRAIL, mediate also some important safeguard mechanisms against tumor growth in patients with CD30+ cutaneous anaplastic large T-cell lymphomas and critically contribute to lymphocyte homeostasis. PMID:21572802

  8. Apoptotic pathway induced by transduction of RUNX3 in the human gastric carcinoma cell line MKN-1.

    PubMed

    Nagahama, Yumi; Ishimaru, Mika; Osaki, Mitsuhiko; Inoue, Toshiaki; Maeda, Akihiro; Nakada, Chisato; Moriyama, Masatsugu; Sato, Kenzo; Oshimura, Mitsuo; Ito, Hisao

    2008-01-01

    The human runt-related transcription factor 3 gene (RUNX3) is considered to be a candidate tumor suppressor gene in gastric carcinoma. However, the role of RUNX3 in the regulation of cell proliferation remains unclear. In the present study, we constructed an adenoviral vector encoding human RUNX3 cDNA under the control of a Tet-responsive promoter (Ad-Tet-FLAG-RUNX3), which regulates the expression of RUNX3 in the presence or absence of doxycycline. A recombinant adenoviral expression vector encoding LacZ (Ad-Tet-LacZ) was used as a negative control. The effect of the transduction of RUNX3 on cell growth was examined using the Tet-On system in a human gastric carcinoma cell line, MKN-1. Exogenous RUNX3 expression was induced successfully by Ad-Tet-FLAG-RUNX3, but not Ad-Tet-LacZ, in the presence of doxycycline in the MKN-1 cells. At 72 h after infection, the proliferative activity in RUNX3-expressing cells was 55% or less of that of the control cells. Flow cytometry revealed that the sub-G(1) peak was increased in cells expressing RUNX3 (34.11%), indicating that the inhibition of cell growth was due to apoptosis, which was confirmed based on Hoechst 33258 staining, the release of cytochrome c from mitochondria into the cytosol, and detection of cleaved caspase-3 by western blotting in MKN-1 cells. Comprehensive analysis using a cDNA microarray showed that RUNX3 upregulated 17 apoptosis-related genes (including FADD, TRAF6, caspase-2, ING1, ING4, Calpain 10, and DNase1) and downregulated 135 apoptosis-related genes (including FLIP, PEA15, TXN2, HSPD1, IKK, and TIAL1) in MKN-1 cells. Pathway analyses to generate functional networks of the genes suggested that promotion of the formation of the death-inducing signaling complex and activation of the mitochondria-mediated pathway were associated with RUNX3-induced apoptosis. In conclusion, our findings suggest that exogenous RUNX3 expression suppressed cell proliferation by inducing apoptosis via the death

  9. Suppression of CSN5 promotes the apoptosis of gastric cancer cells through regulating p53-related apoptotic pathways.

    PubMed

    Sang, Miao-Miao; Du, Wen-Qi; Zhang, Rui-Yan; Zheng, Jun-Nian; Pei, Dong-Sheng

    2015-08-01

    As one of the COP9 signalosome complex, CSN5 (also known as Jab1) has been confirmed overexpression in many human cancers and affected multiple pathways associating with cell proliferation and apoptosis. Correlation of CSN5 overexpression with poor prognosis for cancer provides evidence that it is involved in the tumorigenesis. However, little is known about the functional role and the underlying mechanism of CSN5 in gastric cancer progression. In the current study, the effect of CSN5 siRNA (small-interfering RNA) on the proliferation and apoptosis of human gastric cancer cells (AGS and MKN45) were examined. Our results showed that knockdown of CSN5 could inhibit proliferation and promote apoptosis of gastric cancer cells. Additionally, suppression of CSN5 expression contributed to the increased expression levels of p53 and Bax. In conclusion, CSN5 overexpression is significantly correlated with gastric cancer progression, and CSN5 could be a novel target in gastric cancer therapy.

  10. North American cranberry (Vaccinium macrocarpon) stimulates apoptotic pathways in DU145 human prostate cancer cells in vitro.

    PubMed

    MacLean, Malcolm A; Scott, Bradley E; Deziel, Bob A; Nunnelley, Melissa C; Liberty, Anne M; Gottschall-Pass, Katherine T; Neto, Catherine C; Hurta, Robert A R

    2011-01-01

    Diets rich in fruits and vegetables have been shown to improve patient prognosis in a variety of cancers, a benefit partly derived from phytochemicals, many of which target cell death pathways in tumor cells. Cranberries (Vaccinium macrocarpon) are a phytochemical-rich fruit containing a variety of polyphenolic compounds. As flavonoids have been shown to induce apoptosis in human tumor cells, this study investigated the hypothesis that cranberry-mediated cytotoxicity in DU145 human prostate adenocarcinoma cells involves apoptosis. The results showed that induction of apoptosis in these cells occurred in response to treatment with whole cranberry extract and occurred through caspase-8 mediated cleavage of Bid protein to truncated Bid resulting in cytochrome-C release from the mitochondria. Subsequent activation of caspase-9 ultimately resulted in cell death as characterized by DNA fragmentation. Increased Par-4 protein expression was observed, and this is suggested to be at least partly responsible for caspase-8 activation. Proanthocyanidin-enriched and flavonol-enriched fractions of cranberry also increased caspase-8 and caspase-9 activity, suggesting that these compounds play a possible role in apoptosis induction. These findings indicate that cranberry phytochemicals can induce apoptosis in prostate cancer cells in vitro, and these findings further establish the potential value of cranberry phytochemicals as possible agents against prostate cancer.

  11. Cold Atmospheric Plasma Treatment Induces Anti-Proliferative Effects in Prostate Cancer Cells by Redox and Apoptotic Signaling Pathways

    PubMed Central

    Weiss, Martin; Gümbel, Denis; Hanschmann, Eva-Maria; Mandelkow, Robert; Gelbrich, Nadine; Zimmermann, Uwe; Walther, Reinhard; Ekkernkamp, Axel; Sckell, Axel; Kramer, Axel; Burchardt, Martin; Lillig, Christopher H.; Stope, Matthias B.

    2015-01-01

    One of the promising possibilities of the clinical application of cold plasma, so-called cold atmospheric plasma (CAP), is its application on malignant cells and cancer tissue using its anti-neoplastic effects, primarily through the delivery of reactive oxygen and nitrogen species (ROS, RNS). In this study, we investigated the impact of CAP on cellular proliferation and consecutive molecular response mechanisms in established prostate cancer (PC) cell lines. PC cells showed a significantly reduced cell growth following CAP treatment as a result of both an immediate increase of intracellular peroxide levels and through the induction of apoptosis indicated by annexin V assay, TUNEL assay, and the evaluation of changes in nuclear morphology. Notably, co-administration of N-acetylcysteine (NAC) completely neutralized CAP effects by NAC uptake and rapid conversion to glutathione (GSH). Vitamin C could not counteract the CAP induced effects on cell growth. In summary, relatively short treatments with CAP of 10 seconds were sufficient to induce a significant inhibition of cancer proliferation, as observed for the first time in urogenital cancer. Therefore, it is important to understand the mode of CAP related cell death and clarify and optimize CAP as cancer therapy. Increased levels of peroxides can alter redox-regulated signaling pathways and can lead to growth arrest and apoptosis. We assume that the general intracellular redox homeostasis, especially the levels of cellular GSH and peroxidases such as peroxiredoxins affect the outcome of the CAP treatment. PMID:26132846

  12. Goniothalamin induces cell cycle arrest and apoptosis in H400 human oral squamous cell carcinoma: A caspase-dependent mitochondrial-mediated pathway with downregulation of NF-κβ.

    PubMed

    Li, Lim K; Rola, Ali-Saeed; Kaid, Fahme A; Ali, Abdul Manaf; Alabsi, Aied M

    2016-04-01

    Goniothalamin is a natural occurring styryl-lactone compound isolated from Goniothalamus macrophyllus. It had been demonstrated to process promising anticancer activity on various cancer cell lines. However, little study has been carried out on oral cancer. The aim of this study was to determine the cytotoxic effects of goniothalamin against H400 oral cancer cells and its underlying molecular pathways. Results from MTT assay demonstrated that goniothalamin exhibited selective cytotoxicity as well as inhibited cells growth of H400 in dose and time-dependent manner. This was achieved primarily via apoptosis where apoptotic bodies and membrane blebbing were observed using AO/PI and DAPI/Annexin V-FITC fluorescence double staining. In order to understand the apoptosis mechanisms induced by goniothalamin, apoptosis assessment based on mitochondrial membrane potential assay and cytochrome c enzyme-linked immunosorbent assay were carried out. Results demonstrated that the depolarization of mitochondrial transmembrane potential facilitated the release of mitochondrial cytochrome c into cytosol. Caspases assays revealed the activation of initiator caspase-9 and executioner caspase-3/7 in dose-dependent manners. This form of apoptosis was closely associated with the regulation on Bcl-2 family proteins, cell cycle arrest at S phase and inhibition of NF-κβ translocation from cytoplasm to nucleus. Conclusion, goniothalamin has the potential to act as an anticancer agent against human oral squamous cell carcinoma (H400 cells).

  13. Lutein Inhibits the Migration of Retinal Pigment Epithelial Cells via Cytosolic and Mitochondrial Akt Pathways (Lutein Inhibits RPE Cells Migration)

    PubMed Central

    Su, Ching-Chieh; Chan, Chi-Ming; Chen, Han-Min; Wu, Chia-Chun; Hsiao, Chien-Yu; Lee, Pei-Lan; Lin, Victor Chia-Hsiang; Hung, Chi-Feng

    2014-01-01

    During the course of proliferative vitreoretinopathy (PVR), the retinal pigment epithelium (RPE) cells will de-differentiate, proliferate, and migrate onto the surfaces of the sensory retina. Several studies have shown that platelet-derived growth factor (PDGF) can induce migration of RPE cells via an Akt-related pathway. In this study, the effect of lutein on PDGF-BB-induced RPE cells migration was examined using transwell migration assays and Western blot analyses. We found that both phosphorylation of Akt and mitochondrial translocation of Akt in RPE cells induced by PDGF-BB stimulation were suppressed by lutein. Furthermore, the increased migration observed in RPE cells with overexpressed mitochondrial Akt could also be suppressed by lutein. Our results demonstrate that lutein can inhibit PDGF-BB induced RPE cells migration through the inhibition of both cytoplasmic and mitochondrial Akt activation. PMID:25110866

  14. Hydroxylation of multi-walled carbon nanotubes: Enhanced biocompatibility through reduction of oxidative stress initiated cell membrane damage, cell cycle arrestment and extrinsic apoptotic pathway.

    PubMed

    Liu, Zhenbao; Liu, Yanfei; Peng, Dongming

    2016-10-01

    Modification of CNTs with hydroxyl group promotes their applications in biomedical area. However, the impact of hydroxylation on their biocompatibility is far from being completely understood. In this study, we carried out a comprehensive evaluation of hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) on the human normal liver L02 cell line, and compared it with that of pristine multi-walled carbon nanotubes (p-MWCNTs). Results demonstrated that compared with p-MWCNTs, MWCNTs-OH induced significantly lower oxidative stress as indicated by the level of intracellular antioxidant glutathione (GSH), subsequently lead to less cell membrane damage as demonstrated by lactate dehydrogenase (LDH) leakage assay, and showed slightly decreased arrestment of cell cycle distribution at G0/G1. More interestingly, MWCNTs-OH exhibited significantly lower tendency to activate caspase-8, a key molecule involved in the extrinsic apoptotic pathway. All these in vitro results demonstrated that hydroxylation of MWCNTs enhanced their biocompatibility compare with p-MWCNTs.

  15. Bufalin Inhibits HCT116 Colon Cancer Cells and Its Orthotopic Xenograft Tumor in Mice Model through Genes Related to Apoptotic and PTEN/AKT Pathways

    PubMed Central

    Wang, Jie; Chen, Chao; Wang, Shiying; Zhang, Yong; Yin, Peihao; Gao, Zhongxiang; Xu, Jie; Feng, Dianxu; Zuo, Qinsong; Zhao, Ronghua; Chen, Teng

    2015-01-01

    Aims. To investigate the anticolorectal cancer (CRC) effects of Bufalin, a bioactive polyhydroxysteroid from Venenum Bufonis, using HCT116 human CRC cell and an established orthotopic xenograft model in mice, and to explore the mechanisms of action. Material and Methods. Cultured HCT116 cells or BALB/c mice with orthotopic tumor were treated by Bufalin (positive control: 5-FU). Cell proliferation, apoptosis, and cycling were determined by MTT, Annexin V/PI staining, and flow cytometry, respectively. In mice, tumor inhibition rate and animal survival were calculated. The expressions of PTEN/phosphate-PTEN, AKT/phosphate-AKT, Bad, Bcl-xl, Bax, or Caspase-3 in cells and/or tumors were determined by Western blot or immunohistochemical staining. Results. Bufalin significantly inhibited cell proliferation and induced cell apoptosis and cycle arrest in a dose/time-dependent manner. In the animal model, Bufalin treatment resulted in significant inhibition of tumor growth and prolonged survival. In the Bufalin-treated cultured cells and/or xenograft tumors, the expressions of PTEN, Bad, Bax, and Caspase-3 were significantly increased, while p-AKT and Bcl-xL significantly decreased. Conclusions. Our results indicate that Bufalin inhibit cell proliferation and orthotopic tumor growth by inducing cell apoptosis through the intrinsic apoptotic pathway, which is of pivotal significance in the identification of an anticancer drug that may synergize with Bufalin. PMID:26770191

  16. Camel milk triggers apoptotic signaling pathways in human hepatoma HepG2 and breast cancer MCF7 cell lines through transcriptional mechanism.

    PubMed

    Korashy, Hesham M; Maayah, Zaid H; Abd-Allah, Adel R; El-Kadi, Ayman O S; Alhaider, Abdulqader A

    2012-01-01

    Few published studies have reported the use of crude camel milk in the treatment of stomach infections, tuberculosis and cancer. Yet, little research was conducted on the effect of camel milk on the apoptosis and oxidative stress associated with human cancer. The present study investigated the effect and the underlying mechanisms of camel milk on the proliferation of human cancer cells using an in vitro model of human hepatoma (HepG2) and human breast (MCF7) cancer cells. Our results showed that camel milk, but not bovine milk, significantly inhibited HepG2 and MCF7 cells proliferation through the activation of caspase-3 mRNA and activity levels, and the induction of death receptors in both cell lines. In addition, Camel milk enhanced the expression of oxidative stress markers, heme oxygenase-1 and reactive oxygen species production in both cells. Mechanistically, the increase in caspase-3 mRNA levels by camel milk was completely blocked by the transcriptional inhibitor, actinomycin D; implying that camel milk increased de novo RNA synthesis. Furthermore, Inhibition of the mitogen activated protein kinases differentially modulated the camel milk-induced caspase-3 mRNA levels. Taken together, camel milk inhibited HepG2 and MCF7 cells survival and proliferation through the activation of both the extrinsic and intrinsic apoptotic pathways.

  17. Nitric oxide mediates coral bleaching through an apoptotic-like cell death pathway: evidence from a model sea anemone-dinoflagellate symbiosis.

    PubMed

    Hawkins, Thomas D; Bradley, Benjamin J; Davy, Simon K

    2013-12-01

    Coral bleaching (involving the loss of symbiotic algae from the cnidarian host) is a major threat to coral reefs and appears to be mediated at the cellular level by nitric oxide (NO). In this study, we examined the specific role of NO in bleaching using the sea anemone Aiptasia pulchella, a model system for the study of corals. Exposure of A. pulchella to high-temperature shock (26-33°C over <1 h) or an NO donor (S-nitrosoglutathione) resulted in significant increases in host caspase-like enzyme activity. These responses were reflected in the intensities of bleaching, which were significantly higher in heat- or NO-treated specimens than in controls maintained in seawater at 26°C. Notably, the inhibition of caspase-like activity prevented bleaching even in the presence of an NO donor or at elevated temperature. The additional use of an NO scavenger controlled for effects mediated by agents other than NO. We also exposed A. pulchella to a more ecologically relevant treatment (an increase from 26 to 33°C over 6-7 d). Again, host NO synthesis correlated with the activation of caspase-like enzyme activity. Therefore, we conclude that NO's involvement in cnidarian bleaching arises through the regulation of host apoptotic pathways.

  18. An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis.

    PubMed

    Tao, Weikang; South, Victoria J; Diehl, Ronald E; Davide, Joseph P; Sepp-Lorenzino, Laura; Fraley, Mark E; Arrington, Kenneth L; Lobell, Robert B

    2007-01-01

    The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.

  19. An Inhibitor of the Kinesin Spindle Protein Activates the Intrinsic Apoptotic Pathway Independently of p53 and De Novo Protein Synthesis▿ †

    PubMed Central

    Tao, Weikang; South, Victoria J.; Diehl, Ronald E.; Davide, Joseph P.; Sepp-Lorenzino, Laura; Fraley, Mark E.; Arrington, Kenneth L.; Lobell, Robert B.

    2007-01-01

    The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors. PMID:17101792

  20. Altered of apoptotic markers of both extrinsic and intrinsic pathways induced by hepatitis C virus infection in peripheral blood mononuclear cells

    PubMed Central

    2012-01-01

    Background Chronic hepatitis C (CHC) has emerged as a leading cause of cirrhosis in the U.S. and across the world. To understand the role of apoptotic pathways in hepatitis C virus (HCV) infection, we studied the mRNA and protein expression patterns of apoptosis-related genes in peripheral blood mononuclear cells (PBMC) obtained from patients with HCV infection. Methods The present study included 50 subjects which plasma samples were positive for HCV, but negative for human immunodeficiency virus (HIV) or hepatitis B virus (HBV). These cases were divided into four groups according to METAVIR, a score-based analysis which helps to interpret a liver biopsy according to the degree of inflammation and fibrosis. mRNA expression of the studied genes were analyzed by reverse transcription of quantitative polymerase chain reaction (RT-qPCR) and protein levels, analyzed by ELISA, was also conducted. HCV genotyping was also determined. Results HCV infection increased mRNA expression and protein synthesis of caspase 8 in group 1 by 3 fold and 4 fold, respectively (p < 0.05). In group 4 HCV infection increased mRNA expression and protein synthesis of caspase 9 by 2 fold and 1,5 fold, respectively (p < 0.05). Also, caspase 3 mRNA expression and protein synthesis had level augumented by HCV infection in group 1 by 4 fold and 5 fold, respectively, and in group 4 by 6 fold and 7 fold, respectively (p < 0.05). Conclusions HCV induces alteration at both genomic and protein levels of apoptosis markers involved with extrinsic and intrinsic pathways. PMID:23256595

  1. Grape seed extract targets mitochondrial electron transport chain complex III and induces oxidative and metabolic stress leading to cytoprotective autophagy and apoptotic death in human head and neck cancer cells

    PubMed Central

    Shrotriya, Sangeeta; Deep, Gagan; Lopert, Pamela; Patel, Manisha; Agarwal, Rajesh; Agarwal, Chapla

    2014-01-01

    Head and neck squamous cell carcinoma (HNSCC) is a major killer worldwide and innovative measures are urgently warranted to lower the morbidity and mortality caused by this malignancy. Aberrant redox and metabolic status in HNSCC cells offer a unique opportunity to specifically target cancer cells. Therefore, we investigated the efficacy of grape seed extract (GSE) to target the redox and bioenergetic alterations in HNSCC cells. GSE treatment decreased the mitochondrial electron transport chain complex III activity, increased the mitochondrial superoxide levels and depleted the levels of cellular antioxidant (glutathione), thus resulting in the loss of mitochondrial membrane potential in human HNSCC Detroit 562 and FaDu cells. Polyethylene glycol-SOD addition reversed the GSE-mediated apoptosis without restoring complex III activity. Along with redox changes, GSE inhibited the extracellular acidification rate (representing glycolysis) and oxygen consumption rate (indicating oxidative phosphorylation) leading to metabolic stress in HNSCC cells. Molecular studies revealed that GSE activated AMP-activated protein kinase (AMPK), and suppressed Akt/mTOR/4E-BP1/S6K signaling in both Detroit 562 and FaDu cells. Interestingly, GSE increased the autophagic load specifically in FaDu cells, and autophagy inhibition significantly augmented the apoptosis in these cells. Consistent with in vitro results, in vivo analyses also showed that GSE feeding in nude mice activated AMPK and induced-autophagy in FaDu xenograft tumor tissues. Overall, these findings are innovative as we for the first time showed that GSE targets ETC complex III and induces oxidative and metabolic stress, thereby, causing autophagy and apoptotic death in HNSCC cells. PMID:25557495

  2. SIRT3 in Neural Stem Cells Attenuates Microglia Activation-Induced Oxidative Stress Injury Through Mitochondrial Pathway

    PubMed Central

    Jiang, De-Qi; Wang, Yan; Li, Ming-Xing; Ma, Yan-Jiao; Wang, Yong

    2017-01-01

    Sirtuin 3 (SIRT3), a mitochondrial protein, is involved in energy metabolism, cell apoptosis and mitochondrial function. However, the role of SIRT3 in neural stem cells (NSCs) remains unknown. In previous studies, we found that microglia activation-induced cytotoxicity negatively regulated survival of NSCs, along with mitochondrial dysfunction. The aim of this study was to investigate the potential neuroprotective effects of SIRT3 on the microglia activation-induced oxidative stress injury in NSCs and its possible mechanisms. In the present study, microglia-NSCs co-culture system was used to demonstrate the crosstalk between both cell types. The cytotoxicity of microglia activation by Amyloid-β (Aβ) resulted in the accumulation of reactive oxygen species (ROS) and down-regulation of SIRT3, manganese superoxide dismutase (MnSOD) gene expression in NSCs, concomitant to cell cycle arrest at G0/G1 phase, increased cell apoptosis rate and opening of the mitochondrial permeability transition pore (mPTP) and enhanced mitochondrial membrane potential (ΔΨm) depolarization. Furthermore, SIRT3 knockdown in NSCs via small interfering RNA (siRNA) accelerated cell injury, whereas SIRT3 overexpression provided resistance to microglia activation-induced oxidative stress cellular damage. The mechanisms of SIRT3 attenuated activated microglia-induced NSC dysfunction included the decreased mPTP opening and cyclophilin D (CypD) protein expression, inhibition of mitochondrial cytochrome C (Cyt C) release to cytoplasm, declined Bax/B-cell lymphoma 2 (Bcl-2) ratio and reduced caspase-3/9 activity. Taken together, these data imply that SIRT3 ameliorates microglia activation-induced oxidative stress injury through mitochondrial apoptosis pathway in NSCs, these results may provide a novel intervention target for NSC survival. PMID:28197079

  3. Rapid Eye Movement Sleep Deprivation Induces Neuronal Apoptosis by Noradrenaline Acting on Alpha1 Adrenoceptor and by Triggering Mitochondrial Intrinsic Pathway

    PubMed Central

    Somarajan, Bindu I.; Khanday, Mudasir A.; Mallick, Birendra N.

    2016-01-01

    Many neurodegenerative disorders are associated with rapid eye movement sleep (REMS) loss; however, the mechanism was unknown. As REMS loss elevates noradrenaline (NA) level in the brain as well as induces neuronal apoptosis and degeneration, in this study, we have delineated the intracellular molecular pathway involved in REMS deprivation (REMSD)-associated NA-induced neuronal apoptosis. Rats were REMS deprived for 6 days by the classical flower pot method; suitable controls were conducted and the effects on apoptosis markers evaluated. Further, the role of NA was studied by one, intraperitoneal (i.p.) injection of NA-ergic alpha1 adrenoceptor antagonist prazosin (PRZ) and two, by downregulation of NA synthesis in locus coeruleus (LC) neurons by local microinjection of tyrosine hydroxylase siRNA (TH-siRNA). Immunoblot estimates showed that the expressions of proapoptotic proteins viz. Bcl2-associated death promoter protein, apoptotic protease activating factor-1 (Apaf-1), cytochrome c, caspase9, caspase3 were elevated in the REMS-deprived rat brains, while caspase8 level remained unaffected; PRZ treatment did not allow elevation of these proapoptotic factors. Further, REMSD increased cytochrome c expression, which was prevented if the NA synthesis from the LC neurons was blocked by microinjection of TH-siRNA in vivo into the LC during REMSD in freely moving normal rats. Mitochondrial damage was re-confirmed by transmission electron microscopy, which showed distinctly swollen mitochondria with disintegrated cristae, chromosomal condensation, and clumping along the nuclear membrane, and all these changes were prevented in PRZ-treated rats. Combining findings of this study along with earlier reports, we propose that upon REMSD NA level increases in the brain as the LC, NA-ergic REM-OFF neurons do not cease firing and TH is upregulated in those neurons. This elevated NA acting on alpha1 adrenoceptors damages mitochondria causing release of cytochrome c to activate

  4. Synergism between PGC-1α and estrogen in the survival of endometrial cancer cells via the mitochondrial pathway

    PubMed Central

    Yang, Hui; Yang, Rui; Liu, Hao; Ren, Zhongqian; Kong, Fanfei; Li, Da; Ma, Xiaoxin

    2016-01-01

    Accumulating evidence shows that peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is involved in the progression of hormone-related cancers, and there may exist an association between estrogen and PGC-1α. Notably, emerging evidence has led to considerable interest in the role of PGC-1α in endometrial cancer development. However, whether the synergism exists between PGC-1α and estrogen for regulating mitochondrial function to promote the development of endometrial cancer remains largely unknown. Here, we show that: 1) knockdown of PGC-1α attenuates the survival of endometrial cancer cells by inducing cell apoptosis through the mitochondrial pathway; 2) estrogen remedies the PGC-1α efficiency-induced decline of endometrial cancer cell viability; and 3) estrogen modulates the mitochondrial function to inhibit the PGC-1α deficiency-induced apoptosis in endometrial cancer cells. Collectively, these results demonstrated that the synergism between PGC-1α and estrogen was required for the survival of endometrial cancer cells, which was dependent on the mitochondrial pathway. PMID:27418839

  5. Ethanolic extract of Tulipa edulis Bak induces apoptosis in SGC-7901 human gastric carcinoma cells via the mitochondrial signaling pathway.

    PubMed

    Lin, Ruhui; Li, Zuanfang; Lin, Jiumao; Ye, Jinxia; Cai, Qiaoyan; Chen, Lidian; Peng, Jun

    2015-10-01

    Tulipa edulis Bak (TEB) is an active ingredient in various traditional Chinese medicine compounds and is commonly used to treat swelling and redness, remove toxicity and eliminate stagnation, as well as to prevent and treat certain cancer types. However, the underlying molecular mechanism of the anticancer activity of TEB remains unclear. The aim of the current study was to investigate the effect and underlying mechanism of the ethanolic extract of TEB (EETEB) on SGC-7901 human gastric carcinoma cells. An MTT assay was performed to analyze cell viability. In addition, transmission electron micr